1. UNIVERSITA’
DEGLI
STUDI
DI
ROMA
TOR
VERGATA
FACULTY
OF
MATHEMATICAL,
PHYSICAL
AND
NATURAL
SCIENCES
SINGLE
CYCLE
MASTERS
DEGREE
COURSE
IN
PHARMACY
EXPERIMENTAL
MASTERS
THESIS
IN-­‐VITRO
EVALUATION
OF
ANTIVIRAL
ACTIVITY
OF
NANOFORMULATED
ENFUVIRTIDE
IN
T-­‐
LYMPHOCYTES
SUBMITED
BY:
ZINO
MAMUZO
REFEREES
Professor
FRANCESCA
CECCHERINI-­‐SILBERSTEIN
Professor
CARLO-­‐FEDERICO
PERNO
SUPERVISOR
Dr.
MATTEO
SURDO
Academic Year 2013/2014

2. 2
TABLE OF CONTENTS
ACKNOWLEDGEMT…………………………………………………………………………....3
ABSTRACT……………………………………………………………………………………..…4
CHAPTER 1 INTRODUCTION………………………………………….……………………...5
1.1 HIV
1.1.1.Origin…………………………………………………………..………………..…...6
1.1.2.Classification and genetic distribution of HIV-1……….……………………....…..10
1.1.3.Epidemiology…………………………………………….…………………….…...11
1.1.4.Structure and Morphology……………………………….…………………….…...14
1.1.5.HIV-1 Genome…………………………………………….………………………..16
1.1.6.HIV life Cycle…………………………………………….……...……………..…..22
1.1.7.Pathogenesis of HIV-1 Infection…………………………….…………………..….25
1.1.8.HIV Tropism………………………………………………………..…………...…..27
1.1.9.HIV Co-receptors.…………………………………………………..…………...…..27
1.1.10. HIV Transmission………………………………………………..…………...…..29
1.1.11. Clinical manifestation of HIV-1…………………………………..…………..….30
1.1.12. Diagnosis of HIV………………………………………………..……….…..…...31
1.1.13. Prevention of HIV……………………………………...………..……….…...…..32
1.2 Antiretroviral therapy…………………………………………...………..……….…...…..34
1.2.1 Protease inhibitors (PIs) …………………………………………...………..………...…..37
1.2.2 Reverse transcriptase inhibitors (NRTIs/NNRTIs) ………………...………..………........38
1.2.3 Integrase inhibitors (INIs) ……………………………………...………..……….…...…..40
1.2.4 Entry Inhibitors: Fusion inhibitor (FIs) & CCR5 Antagonist…...………..……….…..…..41
1.2.5 Drug resistance…………………………………………...…………..…..……….…...…..43
1.3 Combination antiretroviral treatment…………………...…………..…..……….…...…..44
1.4 Enfuvirtide a Fusion Inhibitor…………………………...…………..…..……….…....…..46
1.4.1 Mechanism of action…………………………………......…………..…..……….…...…..47
1.4.2 Pharmacokinetics…………………………………...………………..…..……….…...…..48
1.4.2.1 Absorption…………………………………………...…………..…..……….…...…..48
1.4.2.2 Distribution…………………………………………...…………..…..……….…..…..49
1.4.2.3 Metabolism and elimination………………………...…………..…....……….…..…..49
1.4.3 Resistance to Enfuvirtide………………………...………….………..…..……….…..…..50
1.5 Nanotechnology in HIV treatment………………………...…………..…..…………...….51
CHAPTER 2 GENERAL OBJECTIVE AND RATIONAL OF MY WORK..…………....…55
CHAPTER 3 MATERIALS AND METHODS………………...…………..…..…………........56
3.1 Cells used…………………………………………...…………..…..…………....…........56
3.2 Virus used…………………………………………...…………..…..……….…..............57
3.3 Compounds..…………………………………………...…………..…..…………...........57
3.4 Experiments in vitro for the evaluation of antiviral activity of Enfuvirtide………..........58
3.5 Drug toxicity…………………………………………...…………..…..……….…..........60
3.6 Quantification of viral antigen HIV - p24……………...…………..…..……….….........60
CHAPTER 4 RESULTS…………………………………………...…………..…..……….…...62
4.1 Evaluation of antiviral activity of Enfuvirtide and Nano formulated Enfuvirtide…..62
4.2 Evaluation of drug toxicity of Enfuvirtide and its conjugated forms with
FITC (ENF-FITC) and Nanoformulated (MYTS-ENF-FITC) …...............................67
CHAPTER 5 DISCUSSION AND CONCLUSION……………...…………..…..……….…...68
REFERENCES…………………………………………...…………..……….……….…...........73

3. 3
ACKNOWLEDGEMENT
I thank the Blessed Trinity for grace and wisdom in making this project a success despite the
huddles and challenges. Job 23:10, Job 8:7.
Am very grateful to Professor FRANCESCA CECCHERINI-SILBERSTEIN and Professor
CARLO-FEDERICO PERNO, Department of Experimental Medicine and Surgery, University
of Rome “ Tor Vergata” Italy. For granting me this priceless opportunity to be able to carry out
this project in their laboratory. Also for their encouragement, scientific and technical supports
during my six months stay in the laboratory.
I also proceed to thank my supervisor Matteo Surdo for his undisputed supports in this project,
during my stay at the laboratory and his training in the field of experimental virology.
Thanks to Ayele Argaw Denboba, Ogboi Sonny Johnbull, Fejiro, Victor, Debo and Toba for
your scientific advice during this project.
I thank the University of Rome “Tor Vergata,” her School of Pharmacy, my professors,
colleagues and friends for their support and encouragements during my five years stay at the
university.
I thank my lovely family for their supports in every area during my studies. I thank my mummy
especially for her prayers and encouraging words to press on even when all hope was lost. Also
want to thank my elder sister (Fejiro) for her priceless support studying pharmacy together and
her help in making this project a success. Big thanks to my two kid sisters Ufy & Efe for their
love and support. I also want to appreciate my Dad, Cousins, Uncles (Inno, Michael & Godwin)
& Aunties for their kind support during my university studies. Special thanks to my Close Circle
of friends for your love and prayers. To Yemi, Nana, Will, Magda, Ada, Tri, Debola, Pre, Nana
Ama, Anto, Sis Eti, Zoe, Jen & many others, thank you for your support and love during my
studies.
Thank you Samantha for your support over the past Six months and counting, I really appreciate
you.
To my spiritual fathers and mothers, Pst Chris Gold, Pst JRO, Pst Wole, Pst Ruth, and so many
others. To JECALIC members & MFM I say a very big thank you I am very grateful for every
thing you have done for me.
To every one that contributed one way or the other to my studies and this project, God richly
bless you back in return. Galatians 6:9.

4. 4
ABSTRACT
Although current antiretroviral (ARV) therapies suppress plasma HIV below detectable levels in
a consistent proportion of subjects, total virus eradication is still beyond our possibilities. One
important barrier to achieve such goal is related to the suboptimal concentrations of ARVs
within the HIV sanctuaries (HS). HS can be defined as an anatomical (genital tract (GT), gut-
associated lymphoid tissue, lymph nodes (LN), central nervous system (CNS)) or cellular
(macrophages M), latently infected CD4+ T cells) site, impermeable to ARV action and within
which the virus replicates or persists despite treatment. In recent years, different nano-
approaches have been developed to potentiate virus eradication from HS. With regard to the
CNS, nanotechnology could allow ARVs to reach brain HIV-sensitive CD4+ cells, improving
drug permeation across the blood brain barrier (BBB). The blood-testis barrier (BTB) seems to
have a similar role in restricting ARVs penetration into the male GT (MGT) while, in LN, the
unclear suboptimal ARVs concentrations, and the presence of different types of cellular
reservoirs represent an obstacle to HIV eradication. Here, we assess the ability of iron oxide
nanoparticles coated with PMA polymer (MYTS) to act as a delivery system for ARVs to brain,
MGT and LN. The antiretroviral efficacy will be improved by functionalizing nanoparticle with
the anti-CD4 antibody (CD4a). This experiment was performed by using Fusion inhibitor
Enfuvirtide (ENF).

5. 5
CHAPTER 1
INTRODUCTION
1.1 HIV
The Human immunodeficiency virus (HIV), identified in 1983, is a member of the Lentivirus
genus which is exogenous, non-oncogenic retrovirus causing persistent infections leading to
chronic diseases with long incubation periods (lenti for slow). Like the human T-cell leukemia
virus (HTLV) family of primate onco-retrovirus, lentiviruses are complex retrovirus [1]
. The
significant characteristic of the complex retroviruses is the ability to regulate their own
expression via virally encoded protein factors not found in other retroviruses. This property has
been proposed to be essential for the long-term association of the complex retroviruses with the
host and the generation of chronic active infections. The lentiviral complexity is reflected in their
replication cycle, which reveals intricate regulatory pathways, unique mechanisms for viral
persistence [2]
and the ability to infect non-dividing cells.
Retroviruses were traditionally divided into three subfamilies, based mainly on pathogenicity
rather than on genome relationship (oncoviruses which cause neoplastic disorders, spumaviruses
which give cytopathic effect in tissue culture but apparently not associated with any known
disease, and lentiviruses which induce slowly progressing inflammatory, neurological and
immunological diseases). In the last decade, the international committee on the taxonomy of
viruses has recognized seven distinct genera in the Retroviridae family (Table 1.1)[3]

6. 6
Table 1.1. Retroviruses genera
The retrovirus family is divided in 7 genera: the Alpharetroviruses, Betaretroviruses,
Gammaretroviruses, Deltaretroviruses and Epsilonviruses (all of which used to be classified as
one genus, the oncoviruses), the Lentiviruses (which includes HIV) and the Spumaviruses.
1.1.1 Origin
The origin of AIDS and HIV has puzzled scientists ever since the illness first came to light in the
early 1980s. At this time, AIDS did not yet have a name, but it quickly became obvious that all
the men were suffering from a common syndrome. Acquired Immune Deficiency Syndrome
(AIDS) was first recognized as a new disease in 1981 when increasing numbers of young
homosexual men succumbed to unusual opportunistic infections and rare malignancies
[65]
. Kaposi’s sarcoma and Pneumocystis pneumonia among homosexual men—New York City
and California [66]
. A retrovirus, now termed human immunodeficiency virus type 1 (HIV-1), was
subsequently identified as the causative agent of what has since become one of the most
devastating infectious diseases to have emerged in recent history. [67-69]
Causative agent of acquire immunodeficiency syndrome (AIDS).

7. 7
-­‐ 1981- Description of the syndrome in homosexual men features.
Opportunistic infections; KS, Lymphomas and other tumors.
-­‐ 1983 - Isolation of HIV-1
-­‐ 1985 - Genome sequencing
-­‐ 1987 - Isolation of HIV-2

8. 8
Figure 1.1 origin of HIV: http://www.hiv.lanl.gov
There are many schools of thoughts that suggest that HIV-1 and HIV-2 originated from
Primates and were introduced into the human population via cross-species transmission events
[4]
. The viral genome structure of the simian form of the virus, simian immunodeficiency virus
(SIV) is incredibly similar to that of HIV [5]
, and phylogenetic connection has been established

9. 9
[6,7]
. SIV has been shown to infect primates that live in geographical areas wherever HIV is
endemic [6,7]
and attainable routes of transmission, like the butchering of primates for food and
keeping monkeys as pets, are proposed [6]
.
A natural primate host for HIV-1 has been suggested but there still remains some controversy.
Strains of SIV (SIVcpz) from the chimpanzee pan troglodyte’s phylogenetically cluster nearer to
HIV-1 strains than several others characterized SIV strains [6]
. But there's still a moderate
quantity of diversity between SIVcpz and HIV-1, and also the prevalence of SIV infections in
wild chimpanzees is low. HIV-1 is divided in four groups [M (Major), N (non-M/non-O), O
(Outlier) and P (From Gorilla)]. Each of the these groups of HIV-1 share similar branch with
SIVcpz strains, however they do not diverge from a common stem with SIVcpz, suggesting that
they each ascended from completely different cross species transmissions [7,8]
. To support the
concept that P.t. troglodytes is the natural reservoir of HIV, the HIV-1 N cluster was shown to be
a recombinant between various viral strains within the HIV/SIVcpz group, suggesting an
ancestral recombination with this sub-species of chimpanzee [6,9]
.
It is assumed that the HIV-1 M group originated within the Democratic Republic of Congo, as an
extremely high genetic diversity is seen in the region [10]
and also the earliest confirmed HIV-1
infection was found there in a stored serum sample from 1959[11]
. Based on sequence analysis of
the HIV-1 M group, it had been estimated that these strains arose from a common ancestor in
around 1931 [12]
.
Unlike HIV-1, the origin of HIV-2 is more definitive. The discovery of a form of SIV in sooty
mangabeys (SIVsm) that is almost identical to HIV-2, and which is found in these primates from
the area where HIV-2 circulates in humans, provides very robust proof that HIV-2 came from
these primates [13]
. At present, there are eight selected groups of HIV-2, A-H, which are
analogous to the HIV-1 groups (M, N and O) though, groups C-H have solely been identified in
single [14,15]
. All groups of HIV-2 are believed to have arisen from individual cross-species

10. 10
transmission events from sooty mangabeys [16]
. Analysis conducted with HIV-2 strains from
subtypes A and B, dated a recent common ancestor to around 1940 and 1945 respectively [17]
.
1.1.2 Classification and genetic distribution of HIV-1
HIV-1 is widely disseminated worldwide, and can be further divided into genetic groups,
subtypes and sub-subtypes based on the genetic variation and phylogenetic analysis.
HIV-1 strains are divided in four groups [M (Major), N (non-M/non-O), O (Outlier) and P (From
Gorilla)], originating from four separate cross-species transmissions from chimpanzees and/or
gorillas to humans. While HIV-1 groups O, N and P are mainly restricted to Central Africa. HIV-
1 group M is responsible for the AIDS pandemic, accounting for over 90% of HIV infections
worldwide. HIV-1 group M has been further classified into 9 distinct subtypes, A-D, F-H, J and
K subtype and inter-subtype circulating recombinant forms (CRFs). Subtypes and sub-subtypes
arose from founder effects at different time points in the past, and inter-subtype recombinants
can arise in patients co-infected with strains from two different subtypes. If these newly
recombined strains have a significant epidemic spread, they are called Circulating Recombinant
Forms (CRFs).
▪ Subtype A: Central and East Africa as well as East European countries that were
formerly part of the Soviet Union.
▪ Subtype B: West and Central Europe, the Americas, Australia, South America, and
several southeast Asian countries (Thailand, and Japan), as well as northern Africa and
the Middle East.
▪ Subtype C: Sub-Saharan Africa, India, and Brazil.
▪ Subtype D: North Africa and the Middle East.
▪ Subtype F: South and Southeast Asia.

11. 11
▪ Subtype G: West and Central Africa.
Subtypes H, J, and K: Africa and the Middle East.
Figure 1.2a, groups and subtypes of HIV-1.
1.1.3 Epidemiology
Globally, 35.0 million [33.2–37.2 million] people were living with HIV at the end of 2013. An
estimated 0.8% of adults aged 15–49 years worldwide are living with HIV, although the burden
of the epidemic continues to vary considerably between countries and regions. Sub-Saharan
Africa remains most severely affected, with nearly 1 in every 20 adults living with HIV and
accounting for nearly 71% of the people living with HIV worldwide. The Caribbean is the only
other region with an adult HIV prevalence rate above the global average, at 1.0%. Prevalence

12. 12
rates in the remaining regions were all below the global average: 0.7% in Eastern Europe and
Central Asia, 0.5% in North America, 0.4% in Latin America, 0.2% in Oceania, 0.2% in Western
and Central Europe, 0.1% in the Middle East and North Africa, 0.3% in South and South-East
Asia and less than 0.1% in East Asia.
As the acquired immune deficiency syndrome (AIDS) pandemic enters its third decade, the
amount of individuals living with human immunodeficiency virus (HIV) infection continues to
extend. However, the HIV/AIDS epidemic was recognized in Southeast Asia later than
elsewhere, local risk behaviors have allowed the epidemic to expand rapidly. Today, injecting
drug use (IDU) accounts for up to 70th of HIV-1 transmission in several Asian countries,
together with China, Indonesia, Malaysia, Myanmar, eastern India and Vietnam [18]
.
Globally there are two different types of HIV epidemics. In “concentrated” epidemics,
transmission occurs largely in defined vulnerable groups such as sex workers, gay men and other
men who have sex with men, and people who use injection drugs.
In “generalized” epidemics, transmission is sustained by sexual behavior in the general
population and would persist despite effective programs for vulnerable groups. North America
has a concentrated epidemic whereas sub-Saharan Africa has a generalized epidemic. In addition,
there is ample proof that heterosexual transmission through commercial sex workers has
increased over the last few years [18]
.

13. 13
Figure 1.3a Adult HIV worldwide prevalence from age 15-49 in year 2013. [Global Health
Observatory (GHO) data]
Figure
1.3b Global summary of AIDs epidemic in 2013. [UNAIDS 2014]

14. 14
1.1.4 Structure and Morphology
The HIV virion is a spherical virus particle of about 100 nm in diameter (Fig. 1.1). The viral
envelope consists of a lipid bilayer derived from the host cell membrane during release of the
newly produced particles from an infected cell. Embedded in the viral envelope are proteins from
the host cell as well as viral protein complexes composed of the transmenbrane glycoprotein
gp41 (TM) and the surface glycoprotein gp120 (SU). These trimeric TM-SU complexes
constitute the characteristics spike of the virion that is involved in cell recognition and entry.
A matrix shell compromising ca. 2000 copies of the matrix p17 (MA) lines the inner surface of
the viral membrane. In the center of a mature HIV particle resides the cone-shaped capsid (or
cone). The capsid is made of ca. 2000 copies of the viral capsid protein p24 (CA). It encloses
two single strands of the HIV RNA genome stabilized as a ribonucleoprotein complex with ca.
2000 copies of the nucleocapsid protein p7 (NC). Additionally, the capsid contains the three
virally encoded enzymes, reverse transcriptase, protease, and integrase as well as accessory
proteins such as nef, vif, and vpr. There are three additional accessory proteins rev, tat, vpu,
which are not packaged into the virion [58,59]
.

15. 15
Figure 1.4. The immature and mature forms of HIV-1. Typical lentivirus particles are spherical,
about 80-110 nm in diameter, and consist of a lipid bilayer membrane surrounding a conical
core. The two identical single-stranded RNA (ssRNA) molecules, of about 9.2kB each, are
associated with the nucleocapsid proteins p7gag (NC). They are packed into the core along with
virally encoded enzymes: reverse transcriptase (RT), integrase, and protease. P24gag comprises
the inner part of the core, the capsid (CA). The p17gag protein constitutes the matrix (MA),
which is located between the nucleocapsid and the virion envelope. The viral envelope is
produced by the cellular plasma membrane and contains the protruding viral Env glycoproteins:
gp120 surface glycoprotein (SU) and gp41 transmembrane protein (TM).
(from: http://tolomeo.files.wordpress.com/2008/11/hiv.gif )

16. 16
1.1.5 HIV-1 Genome
The genome of HIV has a length of approximately 9.2 kbp. Like all retroviruses it contains the
characteristics: 5‘- gag – pol – env - 3‘ motif consisting of the three structural genes gag, pol,
and env (Fig. 1.5b). The Gag (group antigen) gene encodes the large precursor polyprotein p55
that is cleaved in four proteins: the matrix p17, the "core" capsid p24, the nucleocapsid p7 and
the p6 [19]
. The pol (polymerase) gene encodes the synthesis of three viral enzymes: protease p10,
reverse transcriptase/ribonuclease H complex p51 and p66, integrase p32. The env (envelope)
gene directs the production of an envelope precursor protein gp160, which undergoes cellular
proteolytic cleavage into the outer envelope glycoprotein gp120 and the transmembrane
glycoprotein gp41.
The RNA genome is flanked by two short redundant (R) sequences at both termini with adjacent
unique sequences, U5 and U3, found at the 5‘ and 3‘ ends, respectively. In addition, HIV has at
least six more genes encoding viral proteins with regulatory functions (tat and rev) or accessory
functions (nef, vif, vpr and vpu) (for reviews see [1,60-64]
).
HIV-1 Genomic Composition
The integrated form of HIV-1, also known as the provirus, is approximately 9.5 kb in length.
Both ends of the provirus are flanked by a repeated sequence known as the long terminal repeats
(LTRs). The genes of HIV are located in the central region of the proviral DNA and encode at
least nine proteins. These proteins are divided into three classes:
▪ The major structural proteins, Gag, Pol, and Env
▪ The regulatory proteins, Tat and Rev
▪ The accessory proteins, Vpu, Vpr, Vif, and Nef

17. 17
Figure 1.5a Genetic organization of HIV-1
Figure 1.5b Like all other retroviruses, HIV has three structural genes gag, pol and env , which
are flanked by the long terminal repeats (LTR‘s). In addition it has six more genes, including two
regulatory genes tat and rev and four accessory genes nef, vif, vpr and vpu .

18. 18
Structural Proteins: The major structural proteins are the Gag, Pol, and Env.
GAG
The genomic region encoding the capsid proteins (group specific antigens). The precursor is the
p55 myristoylated protein, which is processed to p17 (Matrix), p24 (Capsid), p7 (NucleoCapsid),
and p6 proteins, by the viral protease. Gag associates with the plasma membrane, where virus
assembly takes place. The 55-kDa Gag precursor is called assemblin to indicate its role in viral
assembly. [81]
Gag-Pol Precursor
The genomic region encoding the viral enzymes protease, reverse transcriptase, and integrase.
These enzymes are produced as a Gag-Pol precursor polyprotein, which is processed by the viral
protease; the Gag-Pol precursor is produced by ribosome frameshifting near the 3' end of gag.
Env
Viral glycoproteins produced as a precursor (gp160), which is processed to give a noncovalent
complex of the external glycoprotein gp120 and the transmembrane glycoprotein gp41. The
mature gp120-gp41 proteins are bound by non-covalent interactions and are associated as a
trimer on the cell surface. A substantial amount of gp120 can be found released in the medium.

19. 19
gp120 contains the binding site for the CD4 receptor, and the seven transmembrane domain
chemokine receptors that serve as co-receptors for HIV-1. [82]
Regulatory Proteins and Accessory Proteins:
In addition to the gag, pol, and env genes contained in all retroviruses, and the tat and rev
regulatory genes, HIV-1 contains four additional genes: nef, vif, vpr and vpu, encoding the so-
called accessory proteins
Table 1.5: overview of the accessory and regulation proteins and their function
Regulatory Proteins:
• TAT
Transactivator of HIV gene expression. One of two essential viral regulatory factors (Tat and
Rev) for HIV gene expression. Two forms are known, Tat-1 exon (minor form) of 72 amino
acids and Tat-2 exon (major form) of 86 amino acids. Low levels of both proteins are found in
persistently infected cells. Tat has been localized primarily in the nucleolus/nucleus by
immunofluorescence. It acts by binding to the TAR RNA element and activating transcription
initiation and elongation from the LTR promoter, preventing the 5' LTR AATAAA
polyadenylation signal from causing premature termination of transcription and polyadenylation.
It is the first eukaryotic transcription factor known to interact with RNA rather than DNA and
Gene Protein Function
tat Tat Transcriptional
activator
that
promotes
synthesis
of
full
length
viral
transcripts
rev Rev Promotes
transport
of
unsliped
and
partially
spliced
mRNA
from
nucleus
to
cytoplam
nef Nef
Nef
down-­‐regulates
CD4
to
avoid
suoerinfection
and
also
down
regulates
MHC
I
to
interfere
with
immune
recogniton
vpu Vpu Facilitate
viral
assembly
and
release
vpr Vpr
facilitates
nuclear
entry
in
nondividing
cells,
and
also
arrests
cells
in
G2/M
phase
of
the
cell
cycle
vif Vif increases
efficeincy
of
infection
and
yield
of
virus

20. 20
may have similarities with prokaryotic anti-termination factors. Extracellular Tat can be found
and can be taken up by cells in culture [83]
.
• REV
The second necessary regulatory factor for HIV expression. A 19-kD phosphoprotein, localized
primarily in the nucleolus/nucleus, Rev acts by binding to RRE and promoting the nuclear
export, stabilization, and utilization of the viral mRNAs containing RRE. Rev is considered the
most functionally conserved regulatory protein of lentiviruses. Rev cycles rapidly between the
nucleus and the cytoplasm.
Accessory Proteins:
VIF
Viral infectivity factor, a basic protein typically 23 kD. Promotes the infectivity but not the
production of viral particles. In the absence of Vif, the produced viral particles are defective,
while the cell-to-cell transmission of virus is not affected significantly. Found in almost all
lentiviruses, Vif is a cytoplasmic protein, existing in both a soluble cytosolic form and a
membrane-associated form. The latter form of Vif is a peripheral membrane protein that is
tightly associated with the cytoplasmic side of cellular membranes.it was discovered that Vif
prevents the action of the cellular APOBEC-3G protein, which deaminates DNA: RNA
heteroduplexes in the cytoplasm [84]
.
VPR
Vpr (viral protein R) is a 96-amino acid (14-kD) protein, which is incorporated into the virion. It
interacts with the p6 Gag part of the Pr55 Gag precursor. Vpr detected in the cell is localized to

21. 21
the nucleus. Proposed functions for Vpr include the targeting the nuclear import of pre-
integration complexes, cell growth arrest, transactivation of cellular genes, and induction of
cellular differentiation. In HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2, and SIV-DRL the Vpx
gene is apparently the result of a Vpr gene duplication event, possibly by recombination.
VPU
Vpu (viral protein U) is unique to HIV-1, SIVcpz (the closest SIV relative of HIV-1), SIV-GSN,
SIV-MUS, SIV-MON and SIV-DEN. There is no similar gene in HIV-2, SIV-SMM, or other
SIVs. Vpu is a 16-kd (81-amino acid) type I integral membrane protein with at least two
different biological functions: (a) degradation of CD4 in the endoplasmic reticulum, and (b)
enhancement of virion release from the plasma membrane of HIV-1-infected cells. Env and Vpu
are expressed from a bicistronic mRNA. Vpu probably possesses an N-terminal hydrophobic
membrane anchor and a hydrophilic moiety. It is phosphorylated by casein kinase II at positions
Ser52 and Ser56. Vpu is involved in Env maturation and is not found in the virion. Vpu has been
found to increase susceptibility of HIV-1 infected cells to Fas killing.
NEF
A multifunctional 27-kd myristoylated protein produced by an ORF located at the 3' end of the
primate lentiviruses. Other forms of Nef are known, including nonmyristoylated variants. Nef is
predominantly cytoplasmic and associated with the plasma membrane via the myristoyl residue
linked to the conserved second amino acid (Gly). Nef has also been identified in the nucleus and
found associated with the cytoskeleton in some experiments. One of the first HIV proteins to be
produced in infected cells; it is the most immunogenic of the accessory proteins. The nef genes
of HIV and SIV are dispensable in vitro, but are essential for efficient viral spread and disease
progression in vivo. Nef is necessary for the maintenance of high viral loads and for the
development of AIDS in macaques, and viruses with defective Nef have been detected in some
HIV-1 infected long term survivors.

22. 22
Nef down regulates CD4, the primary viral receptor, and MHC class I molecules, and these
functions map to different parts of the protein. Nef interacts with components of host cell signal
transduction and clathrin-dependent protein sorting pathways. It increases viral infectivity. Nef
contains PxxP motifs that bind to SH3 domains of a subset of Src kinases and are required for
the enhanced growth of HIV, but not for the down regulation of CD4 [85]
.
1.1.6 HIV-1 Life Cycle
The HIV life cycle consists of multiple steps that involve a complex interaction with a cell
[49,50,51]
. HIV uses the machinery of the CD4 cells to multiply (make copies of itself) and spread
throughout the body. This process is called the HIV life cycle.
The life cycle begins with viral entry, a multi-step interaction between the HIV envelope and the
host target cell surface receptors. In the initial step of HIV entry, the HIV gp120 binds to the host
target cell CD4 receptor, thereby anchoring HIV to the host cell. This interaction generates a
conformational change in the HIV envelope V3 region that stimulates HIV binding with a host
cell coreceptor; the main co-receptors used by HIV are CCR5 and CXCR4. Subsequently, the
viral and host membranes fuse, the viral capsid enters the cell, and the HIV RNA is released.
Once inside the cell, the HIV core dissolves, releasing the two copies of single-stranded HIV
RNA. The next step, referred to as reverse transcription, involves the conversion of the single-
stranded HIV RNA to double-stranded HIV DNA by the HIV enzyme reverse transcriptase. The
reverse transcriptase uses the cellular nucleotides as the building blocks for synthesizing HIV
DNA. Then HIV DNA, which is complexed with other HIV proteins, migrates inside the host
nucleus. The HIV integrase enzyme then catalyzes the integration of the HIV DNA into the host
DNA. Once the HIV DNA has integrated into the host genome, it is referred to as proviral DNA.
The HIV provirus remains part of the host DNA and is perceived by the cell as normal host

23. 23
cellular DNA. The cellular enzymes transcribe the proviral DNA into messenger RNA (mRNA)
and genomic RNA. The control of the transcription of proviral DNA involves multiple factors,
including the HIV Tat protein and cellular modulators. The viral mRNA then is exported out of
the nucleus into the host cell cytoplasm where cellular enzymes translate the viral mRNA into
viral proteins. The larger viral proteins require cleaving into smaller, functional proteins, a step
performed by the HIV enzyme protease. The multiple components of the HIV are then
assembled and as the HIV buds off from the cell, further processing occurs to complete the viral
life cycle, with the final product consisting of a mature HIV virion capable of infecting other
cells.
Likewise, each point in the replication cycle of HIV-1 is a real or potential target for therapeutic
intervention. Thus far, the reverse transcriptase, protease, and integrase enzymes as well as the
process of virus–target cell binding and fusion have proven clinically to be susceptible to
pharmacologic disruption.

24. 24
Figure 1.6. Illustrates the main steps in the HIV-1 replication cycle: binding to the CD4 receptor
and co-receptors; fusion with the host cell membrane; uncoating of the viral capsid; release of
the HIV RNA and proteins into the cytoplasm; reverse transcription of HIV RNA to DNA;
formation of the pre-integration complex (PIC); and translocation into the nucleus. Once in the
nucleus the viral DNA is integrated into the host DNA and subsequently transcribed and
translated to form new viral RNA and viral proteins that translocate to the cell surface to
assemble into new immature virus forms. The new viruses bud off and are released. Finally,
during maturation, the protease enzyme cleaves the structural polyprotein to form mature Gag
proteins, resulting in the production of new infectious virions. The major families of
antiretroviral drugs (green), and the step of the life cycle that they block, are indicated. Also
shown are the key HIV restriction factors (tripartite motif-containing 5α (TRIM5α),
APOBEC3G, SAMHD1 and tetherin; red) and their corresponding viral antagonist (Vif, Vpx and
Vpu; blue). CCR5, CC-chemokine receptor 5; LTR, long terminal repeat; NRTIs, nucleoside
reverse transcriptase inhibitors; NNRTIs, non-nucleoside reverse transcriptase inhibitors.

25. 25
1.1.7 Pathogenesis of HIV-1 Infection
Epidemiologists have long established beyond all reasonable doubt that infection by the human
immunodeficiency virus type 1 (HIV-1) leads to the acquired immune deficiency syndrome
(AIDS). There are several viral and host factors determining the variability in HIV-1 infection
outcome and in rates of disease progression in HIV-1 infected individuals. Cellular tropism,
which defines viral phenotype and receptor co-receptors, which determine viral entry into
various cell types, are the major factors influencing HIV pathogenesis. Despite the intense
research for the last 25 years, the exact mechanism of how these factors contribute to the
dramatic loss of CD4+ T cells and the persistence of R5 and X4 strains during the AIDS status is
still not well identified [34]
. Infection with HIV starts without symptoms or ill feeling and is
accompanied by slight changes in the immune system. This stage spans up to three months after
infection until seroconversion where HIV-specific antibodies can be detected in individuals
following recent exposure. The outcome of infection and duration for disease progression with
clinical symptoms may vary greatly between individuals, but often it progresses fairly slowly [35]
.
It takes several years from primary infection to the development of symptoms of advanced HIV
diseases and immunosuppression. During primary infection, although individuals may look
healthy, the virus is actively replicating in the lymph nodes and blood stream of infected
individuals. As a result, the burst of viral load in their bodies may slowly damage the immune
system [36]
.
Symptomatic stage of disease indicates the late phase of HIV disease (AIDS) where individuals
may be susceptible to other opportunistic infections (OIs)[37]
, such as infections with
Mycobacterium avium, Mycobacterium tuberculosis, Pneumocystis carinii, CMV, toxoplasmosis
and candidiasis. It is agreed that infected individuals develop an AIDS status when their plasma
HIV load is high and the CD4+ T count is less than 200 mm3
.

26. 26
One mechanism HIV weakens the immune system is by infecting and destroying CD4+ T cells,
which in turn leads to immunodeficiency at later stage of disease [38]
. HIV attaches to the CD4+
protein on the surface of these and other cells to gain entry.
The number of CD8+ cytotoxic lymphocytes also decreases and lymphoid cells and tissues are
damaged. However, the presence of CD4+ molecules alone proves to be not enough to allow
viral entry into other cell types such as monocytes and dendritic cells. Therefore, a second
doorway is needed for the virus to gain access to infect cells. This led to the discovery of the
chemokine receptor as essential co-receptors for HIV-1. There are different types of these co-
receptors for different cell types that HIV variants can use for infection of cells. Two main
chemokine receptors have been identified to play a major role in HIV entry, CCR5 and CXCR4
(or fusin).
Figure 1.7: The course of HIV infection and disease. Levels of CD4 and viral load are shown to
correlate with the progress of HIV infection and CD4+ T cell depletion.
(Hassan M. Naif Pathogenesis of HIV-1 infection)

27. 27
1.1.8 HIV Tropism
HIV tropism refers to the cell type that the human immunodeficiency virus (HIV) infects and
replicates in. HIV tropism of a patient's virus is measured by the Trofile assay.
HIV can infect a variety of cells such as CD4+ helper T-cells and macrophages that express the
CD4 molecule on their surface. HIV-1 entry to macrophages and T helper cells is mediated not
only through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on
the target cells but also with its chemokine co-receptors.
Macrophage (M-tropic) strains of HIV-1, or non-syncitia-inducing strains (NSI) use the beta-
chemokine receptor CCR5 for entry and are thus able to replicate in macrophages and CD4+ T-
cells. These strains are now called R5 viruses. [2]
The normal ligands for this receptor—
RANTES, macrophage inflammatory protein (MIP)-1β and MIP-1α—are able to suppress HIV-1
infection in vitro. This CCR5 coreceptor is used by almost all primary HIV-1 isolates regardless
of viral genetic subtype.
T-tropic isolates, or syncitia-inducing (SI) strains replicate in primary CD4+ T-cells as well as in
macrophages and use the alpha-chemokine receptor, CXCR4, for entry. These strains are now
called X4 viruses. The alpha-chemokine SDF-1, a ligand for CXCR4, suppresses replication of
T-tropic HIV-1 isolates. It does this by down-regulating the expression of CXCR4 on the surface
of these cells.
Viruses that use only the CCR5 receptor are termed R5; those that only use CXCR4 are termed
X4, and those that use both, X4R5. However, the use of a coreceptor alone does not explain viral
tropism, as not all R5 viruses are able to use CCR5 on macrophages for a productive infection.
1.1.9 HIV Co-receptors
The discovery of chemokine receptors as essential co-receptors required for HIV entry has to a
large extent rationalized the basis of cellular tropism and better-defined HIV entry into different

28. 28
cells. CCR5 is present on a broad range of cells that can be infected by HIV, including T cells,
monocytes and macrophages. Different HIV strains may be encountered in the body of the
patients, which can be classified into three variants: M-, T- and dual-tropic. Mtropic HIV
variants can infect monocytes, macrophages and T lymphocytes through using CCR5 (R5), but
not T cell lines as these express primarily CXCR4. T-tropic variants, which use CXCR4 (X4) as
their principal coreceptor readily, infect T-cell lines and T lymphocytes but not macrophages [39]
.
Dual-tropic strains of HIV-1 utilize CCR5 and CXCR4 (R5X4) to enter macrophages and T cell
lines, respectively, but they can also utilize combinations of major and minor co-receptors.
Furthermore, there is co-expression of chemokine receptors, especially CCR5 and CXCR4, on
most cell types, including blood and tissue macrophages, dendritic cells and T lymphocytes [40]
.
Some primary, but not laboratory adapted X4; T-tropic isolates can also enter macrophages via
CXCR4 (dual-tropic X4 strains)[41]
. Major and minor co-receptors are also Co-expressed, and
CCR3, in addition to CCR5, can be utilized by M-tropic strains in fetal microglial cells, although
there is no consistent agreement as to the relative importance of CCR3 and CCR5 in adult
microglial cells [42]
.
Thus the current classification of cellular tropism of HIV-1 relies on the differential expression
of CCR5 and CXCR4 in monocytes/ macrophages and T-cell lines [43,44]
. CCR5 is important for
NSI M-tropic strains (R5NSI) of HIV, most commonly observed in early stage of infection. On
the other hand, CXCR4 mostly is associated with SI strains (X4SI), which are more pathogenic,
and they appear in some individuals with more aggressive disease. The dual-tropic HIV-1
variants infect both monocytes/ macrophage and T-cell lines either through CCR5 or CXCR4
(R5X4) respectively.
Other chemokine receptors, principally CCR3 and CCR2b, function as minor HIV co-receptors
[45]
. Co-receptors have also shown to mediate the entry of simian immunodeficiency virus and

29. 29
some M-tropic HIV-1 and HIV-2 strains, including Bonzo/STRL33, Bob/GPR15, US28, CCR8,
CX3CR1/V28, and CCR9 (APJ) [45]
.
Another coreceptor, GPR1, mediates the entry of SIV but not HIV-1
Figure 1.8: Coreceptor usage determines viral entry into different cell types and uncovered the
mystery of cellular tropism. Macrophages and primary T lymphocytes express CCR5 and
CXCR4 where T cell lines express only CXCR4. Macrophage (M)-tropic HIV-1 strains infect
macrophages and lymphocyte using CCR5, while T-cell line (T)-tropic strains infect
lymphocytes and T cell lines (but not macrophages) by using CXCR4. All three-cell types are
infectable by the dual-tropic strains by using either co-receptors for entry. T lymphocytes are
infectable by all strains of HIV. (Hassan M. Naif Pathogenesis of HIV-1 infection)
1.1.10 HIV Transmission
HIV is transmitted when the virus enters the body, usually by injecting infected cells or semen.
The virus can enter in several possible ways. HIV is spread when blood, semen, or vaginal fluids
from an infected person enter another person's body, usually through:

30. 30
▪ Sexual contact. The virus may enter the body through a tear in the lining of the rectum,
vagina, urethra, or mouth. Most cases of HIV are spread this way.
▪ Infected blood. HIV can be spread when a person: Undergoes blood transfusion. Shares
needles, syringes, cookers, cotton, cocaine spoons, or eyedroppers used for injecting
drugs or steroids. When someone is accidentally stuck with a needle or other sharp item
that is contaminated with HIV.
HIV may be spread more easily in the early stage of infection and again later, when symptoms of
HIV-related illness develop.
Most commonly, having sex with an infected partner spreads HIV infection. The virus can enter
the body through the lining of the vagina, vulva, penis, rectum, or mouth during sex. Although
intercourse is the primary risk factor, oral sex transmission is also possible.
• A woman who is infected with HIV can spread the virus to her baby during pregnancy,
delivery, or breast-feeding.
The virus doesn't survive well outside the body, so HIV cannot be spread through casual contact
with an infected person, such as by sharing drinking glasses, by casual kissing, or by coming into
contact with the person's sweat or urine.
1.1.11 Clinical manifestation of HIV-1
Progression from HIV infection to disease is often insidious, but once sufficient immunologic
damage and immunosuppression have occurred, a variety of signs and symptoms appear,
depending on the clinical severity and immunopathology of the disease.
The course of infection can be divided into an acute, an asymptomatic, and symptomatic phase.
The acute phase accounts for the first 5-10 weeks of infection and is characterized by high virus

31. 31
production, and activation of lymphocytes in lymphonodes. Up to 5x103 infectious particles per
ml of blood plasma may be found in the first days after infection. This viremia is curtailed within
a few weeks and level off at the beginning of the asymptomatic phase to the so-called virological
set point that is a predictor of disease progression. During this CD4+ cells numbers decrease at a
low steady rate, while virus replication remains constant at a low rate. The duration of the
asymptomatic phase may last between 2 and 20 years. The end stage of disease, when the patient
develops AIDS, is characterized by CD4+ cells count below 200 copies/ml and increased
quantities of the virus. The number of CD8+ cytotoxic lymphocytes also decreases and lymphoid
cells and tissues are damaged.
1.1.12 Diagnosis of HIV
HIV infection is commonly diagnosed by blood tests. There are three main types of tests that are
commonly used: (1) HIV antibody tests, (2) RNA tests, and (3) a combination test that detects
both antibodies and a piece of the virus called the p24 protein. In addition, a blood test known as
a Western blot is used to confirm the diagnosis.
No test is perfect. Tests may be falsely positive or falsely negative. For example, it can take
some time for the immune system to produce enough antibodies for the antibody test to turn
positive. This time period is commonly referred to as the "testing window period" and may last
six weeks to three months following infection. Therefore, if the initial antibody test is negative, a
repeat test should be performed three months later. Early testing is crucial because early
treatments for HIV helps people avoid or minimize complications. Furthermore, high-risk
behaviors can be avoided, thus preventing the spread of the virus to others.
Most commonly, blood is drawn for an enzyme immunoassay (EIA) or enzyme-linked
immunosorbent assay (ELISA). The test is usually run in a local laboratory, so results can take
one to three days to come back.

32. 32
Other tests can detect antibodies in body fluids other than blood such as saliva, urine, and
vaginal secretions. Some of these are designed to be rapid HIV tests that produce results in
approximately 20 minutes. These tests have accuracy rates similar to traditional blood tests.
OraQuick is an at-home test that uses an oral swab to detect HIV antibodies in oral fluid.
Clearview is another rapid HIV test that can detect HIV antibodies in blood or plasma.
All HIV-positive antibody-screening tests must be confirmed with a follow-up blood test called
the Western blot to make a positive diagnosis. If the antibody test and the Western blot are both
positive, the likelihood of a person being HIV infected is >99%. Sometimes, the Western blot is
"indeterminate," meaning that it is neither positive nor negative. In these cases, the tests are
usually repeated at a later date. In addition, an RNA test for the virus might be done.
The HIV combination test can detect both HIV antibodies and a part of the virus called the p24
protein. Because the p24 protein is present in the blood before the body forms antibodies, this
test may decrease the "window period" and allow for earlier detection of HIV infections.
RNA tests detect HIV RNA in the blood (the viral load). It is not commonly used for screening
but can be helpful in detecting early HIV infection when a person is in the window period.
1.1.13 HIV Prevention
There are a number of ways to prevent and reduce the risk of HIV transmission.
HIV prevention methods try to address the three main routes of transmission listed above.
-­‐ Knowing the facts about HIV and being aware of individual status (HIV-positive or HIV-
negative) makes it easier to prevent HIV infection.
-­‐ HIV awareness education, HIV testing and counseling.

33. 33
Preventing the sexual transmission of HIV
• Condom use (including female condoms), Safer sex education, Treating sexually
transmitted infections, Male circumcision.
Preventing HIV transmission through blood
• Screening blood products, Reducing needle sharing and Stopping needle-stick accidents.
Preventing mother-to-child transmission:
There are a number of steps to preventing mother-to-child transmission of HIV:
• Testing the mother for HIV at their first antenatal appointment, during their third
trimester and after delivery of their baby.
• Treatment should be offered if the mother tests positive.
The baby should be tested when it is born and also offered treatment if positive.
Increasingly, antiretroviral treatment is being used to prevent HIV transmission. Good adherence
to antiretroviral treatment can lower a person’s viral load and reduce the risk of onwards HIV
transmission to others.
Post-exposure prophylaxis (PEP) - emergency HIV treatment:
Emergency treatment to prevent HIV infection, known as post-exposure prophylaxis (PEP), is a
series of antiretroviral drugs taken after potential exposure to HIV.
As of 2013, the prevention regimen recommended in the United States consists of three
medications—tenofovir, emtricitabine and raltegravir—as this may reduce the risk further.
Pre-exposure prophylaxis (PrEP)
HIV treatment known as pre-exposure prophylaxis (PrEP) can be taken before potential exposure
to HIV. For example, if one partner in a relationship is HIV-positive and the other is HIV-
negative (known as a serodifferent couple), the negative partner can take PrEP to protect
themselves from HIV transmission with a daily dose of the medications tenofovir, with or

34. 34
without emtricitabine, is effective in a number of groups including men who have sex with men,
couples where one is HIV positive, and young heterosexuals in Africa.
Vaccines
Researchers are still looking for a vaccine that would offer a significant degree of protection
against HIV infection.
1.2 Antiretroviral Therapy
The drugs currently used to treat HIV-1 infection are directed against the four viral enzymes, an
envelope glycoprotein and a human receptor: protease (PR), reverse transcriptase (RT), the
transmembrane glycoprotein gp41 and more recently, also against Integrase (IN) and human
CCR5 receptors. In figure 1.10 all anti-HIV compounds currently approved for clinical use by

35. 35
the U.S. Food and Drug Administration (FDA) [Division of AIDS, National Institute of Allergy
and Infectious Diseases, National Institutes of Health] as of February 2015 are listed. In figure
1.9 the available drugs in clinic by today, according with the viral life cycle steps impaired are
shown.
Figure 1.9 Schematic description of the mechanism of the six classes of currently available
antiretroviral drugs against HIV.

36. 36
Initial entry of HIV into a target cell can be blocked by use of the entry inhibitor maraviroc,
which prevents viral interaction with the CCR5 coreceptor. Fusion of the viral membrane with
the target cell membrane can be blocked by the peptidic inhibitor enfuvirtide, which prevents a
conformational change in the viral Env protein needed to bring the two membranes into close
proximity. Reverse transcription of the viral RNA into DNA can be blocked by nucleoside/tide
reverse transcriptase inhibitors (NRTIs), which are incorporated into the viral DNA, and act to
chain terminate DNA synthesis. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are
non-competitive inhibitors of reverse transcriptase. Integrase strand transfer inhibitors (INSTIs),
such as raltegravir, are active site inhibitors of the viral integrase enzyme and prevent the strand
transfer reaction, the final ligation of the 3_-processed viral DNA into the host genome. Protease
inhibitors (PIs) prevent the proteolytic processing of translated viral proteins by the viral
protease enzyme, resulting in defective virions. Combinations of drugs from two or more of
these classes when combined together form the basis of highly active antiretroviral therapy
(HAART).

37. 37
Figure 1.10.This graphic shows the FDA-approved antiretroviral medications as of March 2015
for use in the United States.
1.2.1 Protease Inhibitors (PIs)
The HIV PIs selectively bind to and inhibit HIV protease, an enzyme that cleaves viral
polyprotein precursors into individual functional proteins. The HIV protease normally cleaves
proteins from the Gag-Pol polyprotein precursor, forming functional smaller proteins in the
process. If the PI successfully blocks the HIV protease, deformed HIV particles are formed and
these particles have diminished infectious capacity [52]
. There are 10 PIs that have been FDA-
approved (Figure 1.11): amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir-
ritonavir, nelfinavir, ritonavir, saquinavir hard gel capsule (Invirase) and saquinavir soft gel
capsule (Fortovase), and tipranavir. Most PIs are now used in combination with low-dose
ritonavir, with the ritonavir acting as a pharmacokinetic booster by inhibiting the metabolism of
other PIs [53]
.

38. 38
1.2.2 Reverse transcriptase inhibitors (NRTIs/NNRTIs)
Ø NUCLEOSIDE AND NUCLEOTIDE REVERSE TRANSCRIPTASE
INHIBITORS.
The NRTIs and NtRTIs target the step of HIV reverse transcription [54]
. To reach their active
form, the NRTIs must undergo three phosphorylation steps within the cell to reach the active
triphosphorylated state; the NtRTIs require only two phosphorylation steps. After the NRTIs (or
NtRTI) reach a triphosphorylated state they structurally resemble the cellular nucleotides and the
HIV reverse transcriptase mistakenly incorporates the drug into the elongating strand of viral

39. 39
DNA. Once incorporated into viral DNA, the NRTI (or NtRTI) has a caboose-like effect by
acting as a chain terminator, thus preventing further chain linkages in the elongating strand of
viral DNA. There are eight FDA-approved drugs in this class (Figure 1.12). Abacavir,
didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, and zidovudine. The
drug zalcitabine is no longer manufactured. The reverse transcriptase inhibitor tenofovir is
classified as a NtRTI. In addition, there are four fixed-drug reverse transcriptase inhibitor
formulations: abacavir-lamivudine (Epzicom), tenofovir-emtricitabine (Truvada), zidovudine-
lamivudine (Combivir), and zidovudine-lamivudine-abacavir (Trizivir).
Ø Non-Nucleoside Reverse Transcriptase Inhibitors
The mechanism of action of the NNRTIs is distinct from the NRTIs: the NNRTIs do not become
incorporated into viral DNA, but instead directly bind to the hydrophobic pocket located close to
the catalytic domain of the reverse transcriptase enzyme complex, thereby preventing the normal

40. 40
dynamic movement of the enzyme complex. In addition, unlike the NRTIs, drugs in the NNRTI
class do not require phosphorylation to become activated. There are five FDA-approved NNRTIs
(Figure 1.13): delavirdine, efavirenz, etravirine, nevirapine, and Rilpivirine. In addition, the
fixed-drug combination pills are tenofovir-emtricitabine-efavirenz (Atripla) and tenofovir-
emtricitabine-rilpivirine (Complera). Efavirenz is not recommended for women with child
bearing potential if they are not using effective contraception. The NNRTIs delavirdine,
efavirenz, and nevirapine are not active against HIV-2; etravirine and rilpivirine have limited
activity against HIV-2.
1.2.3 Integrase Strand Transfer Inhibitors (INSTI)
The integrase strand transfer inhibitors interfere with the insertion of HIV DNA into host DNA
[55]
. The integration of HIV into host DNA is a complex process involving six major steps: (1)
HIV integrase binds to the HIV DNA; (2) HIV integrase catalyzes the 3' processing of the ends

41. 41
of the HIV DNA; (3) the HIV DNA, complexed with integrase and other HIV proteins, migrates
inside of the host nucleus through the nuclear pores; (4) the HIV DNA-protein complex binds to
the host DNA; (5) HIV integrase catalyzes the strand transfer of the HIV DNA into the host
DNA; and (6) human enzymes repair the gaps left following the stand transfer process. Three
integrase strand transfer inhibitors are FDA approved: dolutegravir, elvitegravir and raltegravir.
(Figure 1.14)
1.2.4 Entry Inhibitors (EIs)
The entry inhibitor class now includes two subclasses: (1) CCR5 co-receptor antagonists and (2)
fusion inhibitors, with one FDA-approved drug from each subclass (Figure 1.15)[56]
. In the
process of viral entry (after binding to the host CD4 receptor) HIV can potentially bind to either

42. 42
the host CCR5 or CXCR4 coreceptor. The HIV coreceptor binding depends on the HIV subtype:
the HIV subtype known as R5 HIV (or CCR5-tropic HIV) preferentially binds to the CCR5
coreceptor whereas the X4 HIV (or subtype CXCR4-tropic HIV) binds to the CXCR4
coreceptor. Those strains of HIV that can enter via either the CCR5 or CXCR4 coreceptor are
know as R5X4 HIV (or dual-tropic HIV). Patients with a detectable mixture of R5 and X4 HIV
are considered to have mixed-tropic HIV. The CCR5 antagonists exert their mechanism of action
by binding to the CCR5 coreceptor, causing a conformational change in the coreceptor that
prevents the HIV gp120 from binding with the CCR5 coreceptor. The drug maraviroc
(Selzentry) is the only FDA-approved CCR5 inhibitor and is recommended for use in
antiretroviral treatment-experienced patients who have R5 HIV (CCR5-tropic HIV)[57]
. Prior to
starting a patient on maraviroc, an HIV Tropism Assay or envelope genotype should be
performed to document that the patient has R5 (CCR5-tropic) HIV. The fusion inhibitor
enfuvirtide is a 36-amino acid synthetic peptide that corresponds with a segment in the HIV gp41
known as the heptad repeat region 2. In the normal fusion process, the HIV gp41 heptad repeats
region 2 folds back on the heptad repeat region 1, in essence zipping up the gp41. This process
pulls the HIV and host membranes together and results in the fusion of the viral and host
membranes. The enfuvirtide peptide works by binding to the hepatad repeat region 1, thus
preventing the normal interaction and folding of the gp41 heptad repeat regions 1 and 2.
Enfuvirtide is the only FDA-approved fusion inhibitor and it is indicated in antiretroviral
treatment-experienced patients. The drug enfuvirtide is not active against HIV-2.

43. 43
1.2.5 Drug resistance
Resistance is the cause and/or the consequence of treatment failure. HIV infection is
characterized by a very high replication rate, with the production of 1 to 10 billion new virus
particles per day in an untreated infected individual [20]
. Moreover, HIV-1 RT lacks
exonucleolytic proof-reading functionality, and this results in an average error rate per detectable
nucleotide incorporated of 1/1700 [21]
. Combining these two factors with the length of the viral
genome (∼10,000 nucleotides), it can be calculated that a mutant at each nucleotide position in
the viral genome is produced every day. As a consequence, suboptimal treatments, like
monotherapy regimens, will readily select the mutants in the replicating population that are
resistant to the administered drug(s). Moreover, the selected drug resistant viruses will
compromise the efficacy of subsequent HAART regimens, as extensive cross-resistance was
rapidly observed within each class of antiretroviral drugs.

44. 44
1.3 Combination antiretroviral treatment
Current HAART options are combinations (or "cocktails") consisting of at least three
medications belonging to at least two types, or "classes," of antiretroviral agents. Initially
treatment is typically a non-nucleoside reverse transcriptase inhibitor (NNRTI) plus two
nucleoside analogue reverse transcriptase inhibitors (NRTIs). Typical NRTIs include: zidovudine
(AZT) or tenofovir (TDF) and lamivudine (3TC) or emtricitabine (FTC). Combinations of
agents which include a protease inhibitors (PI) are used if the above regimen loses effectiveness.
High rate of viral replication, low fidelity of reverse transcription, and the ability to recombine
are the viral characteristics that lead to the diversity of HIV-1 species (quasi-species) in
chronically infected individuals. This high genetic variability provided the rationale for highly
active antiretroviral treatments (HAART). By combination of several potent antiretroviral
agents, viral replication is suppressed to such low levels that emergence of drug resistant HIV-1
variants was, if not prevented, at least delayed. By doing so, CD4+ T-lymphocyte numbers

45. 45
increase, leading to a degree of immune reconstitution that is sufficient to reverse clinically
apparent immunodeficiency. Widespread introduction of HAART in industrialized countries
resulted in a striking decrease in morbidity and mortality, putting forward the hope that HIV-1
infection can be transformed into a treatable chronic disease.
A set of criteria composed of plasma viraemia concentration, absolute or relative CD4+ cell
counts, and clinical manifestations, is used to recommend initiation of HAART. The benefits of
treatment clearly outweigh the potential side effects in patients with clinical signs of
immunodeficiency (e.g., AIDS defining illnesses) or with CD4+ numbers less than 200 per µL.
However, the best time point to begin treatment remains controversial in asymptomatic patients
with modest depletion of CD4+ T cells (e.g., more than 350 per µL) and modest levels of
viraemia (e.g., less than 100 000 copies per mL)
Benefits of treatment include a decreased risk of progression to AIDS and a decreased risk of
death. In the developing world treatment also improves physical and mental health. With
treatment there is a 70% reduced risk of acquiring tuberculosis. Additional benefits include a
decreased risk of transmission of the disease to sexual partners and a decrease in mother-to-child
transmission. The effectiveness of treatment depends to a large part on compliance.

46. 46
1.4 Enfuvirtide, a Fusion Inhibitor
This drug interferes with viral entry into the host cell by disrupting the interaction between the
viral glycoprotein gp41 and the target cell membrane [22]
.
The entry of human immunodeficiency virus type 1 (HIV-1) into target cells requires a fusion
reaction of viral and cellular membranes. This process is mediated by the viral envelope
glycoprotein (Env), a trimeric complex consisting of three transmembrane gp41 subunits and
three non-covalently attached gp120 surface subunits. The gp120 subunit is responsible for
attachment to the target cells that initiates the entry process by binding to the CD4 receptor and a
co-receptor (CCR5 or CXCR4), while the gp41 subunit mediates the membrane fusion to deliver
the viral genetic material into target cells. Structurally, the fusion protein gp41 can be divided
into multiple functional domains: a hydrophobic fusion peptide (FP) at the N-terminus, a fusion
peptide proximal region (FPPR), a N-terminal heptad repeat (NHR), a loop with a disulfide bond
at its basis, a C-terminal heptad repeat (CHR), a membrane proximal external region (MPER), a
transmembrane domain (TM), and a long cytoplasmic tail.
The virus-receptor binding can trigger large conformational changes in both gp120 and gp41,
and subsequently activates the fusion machinery of gp41. In the current model, there are at least
three conformational changes in the gp41 ectodomain involved in membranes fusion. The first
conformational change is its metastable native state on the virion surface, which is sheltered by
gp120. The second one is its transition from the native state into the pre-hairpin intermediate
state that releases the hydrophobic gp41 ectodomain in extended conformation, and thus the
fusion peptide inserts into cell membrane. The third is refolding of gp41 into a low energy
trimeric hairpin driven by the NHR and CHR. Cystallographic studies determined the core
structure of hairpin as a six-helical bundle (6-HB), in which three NHR form a central trimeric
coiled coil, whereas three CHR around the NHR pack as antiparallel helices into the hydrophobic

47. 47
grooves. This conformation brings the viral and cellular membranes into close proximity for
efficient fusion.
Figure 1.16. Enfuvirtide (ENF/Fuzeon/T-20)
1.4.1 Mechanism of Action
HIV entry into cells expressing CD4 receptors (e.g. T cells, macrophages) is a complex,
multistep process that involves a series of interactions between the viral envelope glycoprotein
and human cellular receptors. The HIV-1 viral envelope is made up of many subunits, each of
which consists of a gp120 surface protein noncovalently attached to a gp41 transmembrane
protein. The process of HIV entry begins with attachment of gp120 to CD4 receptors on the host
cell. Although binding to CD4 is considered crucial, a chemokine coreceptor, CCR5 or CXCR4,
is also necessary for entry. This binding of gp120 to CD4 induces conformational changes and
structural rearrangements in gp120, exposing the coreceptor-binding site and allowing
interaction with a chemokine coreceptor (CCR5 or CXCR4) on the surface of the host cell. The
attachment of gp120 to both the CD4 and chemokine receptors then triggers key fusogenic
conformational changes in gp41, including exposure of a fusion peptide that inserts into the
plasma membrane of the host cell to bring about the fusion process. The envelope glycoprotein
gp41 is a trimeric structure consisting of 3 gp41 oligomers.

48. 48
Figure 1.17. Initial interaction between gp120 and CD4. 2. Conformational change in gp120
allows for secondary interaction with CCR5. 3. The distal tips of gp41 are inserted in to the
cellular membrane. 4. gp41 undergoes significant conformational change; folding in half and
forming coiled-coils. This process pulls the viral and cellular membranes together, fusing them.
1.4.2 Pharmacokinetics
The pharmacokinetic properties of enfuvirtide were evaluated in HIV-1 infected adult and
pediatric subjects.
1.4.2.1 Absorption
Following a 90-mg single subcutaneous injection of FUZEON into the abdomen in 12 HIV-1
infected subjects, the mean (±SD) Cmax was 4.59 ± 1.5 µg/mL, AUC was 55.8 ± 12.1 µg·h/mL
and the median Tmax was 8 hours (ranged from 3 to 12 h). The absolute bioavailability (using a
90-mg intravenous dose as a reference) was 84.3% ± 15.5%. Following 90-mg twice daily
dosing of FUZEON subcutaneously in combination with other antiretroviral agents in 11 HIV-1
infected subjects, the mean (±SD) steady-state Cmax was 5.0 ± 1.7 µg/mL, Ctrough was 3.3 ±

49. 49
1.6 µg/mL, AUC0-12h was 48.7 ± 19.1 µg·h/mL, and the median Tmax was 4 hours (ranged
from 4 to 8 h).
Absorption of the 90-mg dose was comparable when injected into the subcutaneous tissue of the
abdomen, thigh or arm.
1.4.2.2 Distribution
The mean (±SD) steady-state volume of distribution after intravenous administration of a 90-mg
dose of FUZEON (N=12) was 5.5 ± 1.1 L.
Enfuvirtide is approximately 92% bound to plasma proteins in HIV-infected plasma over a
concentration range of 2 to 10 µg/mL. It is bound predominantly to albumin and to a lower
extent to α-1 acid glycoprotein.
The CSF levels of Enfuvirtide (measured from 2 hours to 18 hours after administration of
Enfuvirtide) in 4 HIV-infected subjects were below the limit of quantification (0.025 µg/mL).
1.4.2.3 Metabolism/Elimination
As a peptide, Enfuvirtide is expected to undergo catabolism to its constituent amino acids, with
subsequent recycling of the amino acids in the body pool. Mass balance studies to determine
elimination pathway(s) of Enfuvirtide have not been performed in humans.
In vitro studies with human microsomes and hepatocytes indicate that Enfuvirtide undergoes
hydrolysis to form a deamidated metabolite at the C-terminal phenylalanine residue, M3. The
hydrolysis reaction is not NADPH dependent. The M3 metabolite is detected in human plasma
following administration of Enfuvirtide, with an AUC ranging from 2.4% to 15% of the
Enfuvirtide AUC.
Following a 90-mg single subcutaneous dose of Enfuvirtide (N=12) the mean ±SD elimination
half-life of Enfuvirtide is 3.8 ± 0.6 h and the mean ±SD apparent clearance was 24.8 ± 4.1

50. 50
mL/h/kg. Following 90-mg twice daily dosing of FUZEON subcutaneously in combination with
other antiretroviral agents in 11 HIV-1 infected subjects, the mean ±SD apparent clearance was
30.6 ± 10.6 mL/h/kg.
1.4.3 Resistance to Enfuvirtide
Enfuvirtide resistance mutations have been investigated in patients failing therapy, these
mutations cluster in the HR1 domain where Enfuvirtide binds to gp41 and include G36D, V38A,
N42D, and N43D/Q. Mutations in env outside of HR1 appear to play little role in Enfuvirtide
resistance. While these resistance mutations decrease sensitivity to Enfuvirtide, they are
associated with reduced efficiency of fusion and enhanced susceptibility to neutralizing
antibodies. Compensatory mutations in the HR2 domain of gp41 can restore viral fusion kinetics
while retaining Enfuvirtide resistance. Importantly, patients that continue Enfuvirtide treatment
despite the presence of resistance mutations retain CD4+ T cell increases from therapy
potentially due to antiviral effects under conditions of incomplete virologic suppression [23,24,25]
.
Figure 1.18 Resistance of Enfuvirtide. Wensing AM. Et. al 2014.
Resistance to Enfuvirtide is associated primarily with mutations in the first heptad repeat (HR1)
region of the gp41 envelope gene. However, mutations or polymorphisms in other regions of the
envelope (e.g., the HR2 region or those yet to be identified) as well as coreceptor usage and
density may affect susceptibility to Enfuvirtide.

51. 51
Cross-resistance
HIV-1 clinical isolates resistant to nucleoside analogue reverse transcriptase inhibitors (NRTI),
non-nucleoside analogue reverse transcriptase inhibitors (NNRTI), and protease inhibitors (PI)
were susceptible to Enfuvirtide in cell culture.
1.5 Nanotechnology in HIV treatments:
Suboptimal adherence, toxicity, drug resistance and viral reservoirs make the lifelong treatment
of HIV infection challenging. The emerging field of nanotechnology may play an important role
in addressing these challenges by creating drugs that possess pharmacological advantages arising
out of unique phenomena that occur at the “Nano“ scale. At these dimensions, particles have
physicochemical properties that are distinct from those of bulk materials or single molecules or
atoms.
Nanotechnology entails the synthesis and manipulation of materials or systems where at least
one dimension is in the nanometer range i.e., in the order of billionths (10-9
) of a meter [26–32]
.
Particles in this size range have unique physicochemical properties, which are distinct from those
of bulk materials (the macroscopic or microscopic scale) or single atoms or molecules (the
atomic scale) [27,29,30,32,33]
. When Nano sized particles come into contact with biological systems,
the nature of the interaction is critically influenced by these physicochemical properties.
Furthermore, many biological phenomena, such as immune recognition and passage across
biological barriers, are governed by size considerations. Drugs fabricated at the appropriate Nano
scale dimensions may therefore have certain physicochemical and biological properties that in
turn confer pharmacological advantages when compared to conventional agents. Research in
nanotechnology may translate into benefits for HIV infected patients, particularly if the
challenges associated with HIV and their treatments are addressed.

52. 52
Ensuring that a Nano pharmaceutical (a Nano particle for pharmaceutical use) reaches its target
sites in its active form is a significant challenge in nanotechnology. This challenge may be
addressed by optimizing the physicochemical properties of the Nano pharmaceutical (charge and
size) or by modifying its surface (e.g., biofunctionalization by attachment of ligands or agents
that prevent opsonization, facilitate transport across membranes or enable targeting). Some of the
methods are listed below.
Optimization of the physicochemical properties of a nanoparticle: Size and charge have an
important influence on the stability, bio distribution and efficacy of a nanoparticle.
Depending on its size, a particle may or may not, for example, traverse the endothelial barrier or
be sequestered by the spleen, trapped within lymphatic tissues or cleared by the kidney. The size
of a nanoparticle also determines the mechanism by which it enters the cell, and where in the cell
it localizes. The surface charge of a nanoparticle influences whether or not it traverses the
negatively charged cell membrane. Size also significantly influences the opsonization of
nanoparticles by plasma proteins.
Pegylation: coating them with polyethylene glycol, a process dubbed “pegylation”, which
reduces opsonization, phagocytosis and uptake by the Reticuloendothelial system, may extend
the plasma circulation of particles.
Overcoming the blood brain barrier: Penetration of the blood-brain barrier may be achieved by
attachment of agents to Nano pharmaceuticals that inhibit efflux transporters. Efflux transporters
are responsible for poor penetration of certain antivirals into the brain.
Targeting: Nano pharmaceuticals may, through active or passive targeting, accumulate
preferentially in specific tissues or cells. Passive targeting occurs when nanoparticles (or other
therapeutic or diagnostic agents) leak into diseased tissue due to the enhanced permeability of

53. 53
the local vasculature. The increased leakiness of the vasculature may be due to malignancy or
inflammation and the therapeutic agent therefore achieves its maximum concentration at the site
of disease. In active targeting, ligands attached to a Nano pharmaceutical bind specifically to
receptors or epitopes that are overexpressed in diseased tissues or cells, thereby causing them to
accumulate at the diseased site.
Challenges to widely apply nanomedicine for HIV/AIDS therapy.
Even though, nanomedicines are the promising future of HIV/AIDS prevention and treatment,
several hurdles remain unresolved, including but not limited to toxicity, unwanted biological
interactions and the difficulty and cost of large-scale synthesis of nanopharmaceuticals [46]
.
Another challenge is the fact that targeted delivery of antiretroviral drugs using nano polymers to
viral reservoir sites may lead to HIV drug resistance. This could be because of two reasons.
Firstly, the targeted delivery of an antiretroviral drugs which if not accompanied by systemic
HAART administration will lead to suboptimal doses of the drug in non-targeted tissues, with
the potential to select out drug resistant mutations there. Furthermore, most studies involving
nanocarriers use a single antiretroviral drug, which would effectively select out resistant virus in
targeted tissues [47,48]
. Highlighting the need to combine at least three drugs for use with
nanocarriers as in conventional HAART in future researches.

54. 54
Figure 1.19 Selected drugs currently used in combination antiretroviral therapy and their ability
to reach the central nervous system, as reflected by the cerebrospinal fluid: blood plasma (CSF:
BP) concentration ratio in humans. For Enfuvirtide (highlighted with yellow) see reference
number 87.

55. 55
CHAPTER 2
GENERALE OBJECTIVE AND RATIONAL OF MY WORK
Antiretroviral drug therapy plays a cornerstone role in the treatment of human
immunodeficiency virus (HIV)/acquired immunodeficiency syndrome patients. Despite obvious
advances over the past 3 decades, new approaches toward improved management of infected
individuals are still required. Drug distribution to the central nervous system (CNS) is required in
order to limit and control viral infection, but the presence of natural barrier structures, in
particular the blood–brain barrier, strongly limits the perfusion of anti-HIV compounds into this
anatomical site. Nanotechnology-based approaches may help providing solutions for antiretro-
viral drug delivery to the CNS by potentially prolonging systemic drug circulation, increasing
the crossing and reducing the efflux of active compounds at the blood–brain barrier, and
providing cell/tissue-targeting and intracellular drug delivery.
Targeted delivery to HIV reservoir sites would be of significant benefit because many
antiretroviral drugs do not penetrate these sites optimally which contribute not only to viral
persistence, but also to the development of drug resistance.
Based on the very extreme low CSF levels of Enfuvirtide in HIV-infected subjects (below the
limit of quantification of 0.025 µg/mL), as a proof of concept we used the nanoformulation of
Enfuvirtide in the present study, to test in-vitro its antiviral activity in T-lymphocytes.

56. 56
CHAPTER 3
MATERIALS AND METHODS
3.1 Cells used
C8166
C8166 cell line was used for the in-vitro evaluation of antiviral activity of nanoformulated
Enfuvirtide in T-lymphocytes. C8166 cell line was obtained from the NIH (National Institute of
Health, USA). C8166 are a human CD4+
, CD3+
, T-lymphoblastoid cell line originally derived by
fusion of primary umbilical cord blood cells with HTLV-1 producing line from adult T cell
leukemia lymphoma patient and it grows in suspension in in vitro cell culture.
The cells were maintained in culture using the RPMI1640 culture medium (Roswell Park
Memorial Institute 1640), produced by Gibco. 10% of fetal bovine serum (FBS) was added to
the culture medium, previously heat-inactivated (56°C for thirty minutes), together with
penicillin (100U/ml) and streptomycin (100µg/ml) by EuroClone and finally L-Glutamine
(2mM) by EuroClone. The cells cultures were maintained at 37°C in a humidified atmosphere at
5%CO2. Every three days, the cells were sub cultured with a dilution of 1:3 in the new flask of
25cm2
in a final volume of 7ml.
293T cells
The HEK293T cell line was used for the transfection of pNL4-3 virus. The 293T cell line is a
permanent line established from primary embryonic human kidney transformed with human
adenovirus type 5 DNA. The cells were obtained from Cell Biolabs, Inc.
The cells were maintained in culture using D-MEM (high glucose) manufactured by Gibco. 10%
of fetal bovine serum (FBS) was added to the culture medium, previously heat-inactivated (56 °C
for thirty minutes), together with penicillin (100U/ml) and streptomycin (100µg/ml) by

57. 57
EuroClone, and finally L-Glutamine (2mM) by EuroClone. The cells cultures were maintained at
37°C in a humidified atmosphere at 5% CO2.
Every three days, the growth medium of the cells was carefully removed without touching the
cell surface, and gently washed with PBS. Trypsin-EDTA (1ml) was used to dis-adhere the cells
and place in a 37°C incubator for about 2 minutes for the cells to be released from the flask. An
appropriate volume of culture D-MEM medium was added to re-suspend the cells and inactivate
the trypsin. The cells were cultured in a new culture medium with a dilution of 1:3 in a flask of
25cm2
with a final volume of 7ml and stored at 37°C in a humidified atmosphere at 5% CO2.
3.2 Virus used
The CXCR4 pNL4-3 virus was prepared by transfection. HEK-293T cells were plated in a 6 well
plates (800.000cells/well) 24 hours before transfection. On the day of transfection, cell density
was approximately 70-80% confluent. The pNL4-3 plasmid used for this study was transfected
using the Transit 2020 reagent (TEMA) according to the manufacturer. At three days post-
transfection the virus was harvested, filtered and aliquots stored at -80°C. In order to quantify
viral particles amount, supernatants were analyzed for p24 production by a commercial Elisa kit,
according to the manufacturer (Ag-GenscreenTM
HIV-1 Assay, BioRad).
3.3 Compounds
Enfuvirtide (Fuzeon powder) a fusion inhibitor, synthesized under GMP conditions, was
obtained from Roche; a stock solution, diluted in PBS, was made fresh for each experiment.
Stock solutions of Enfuvirtide conjugated with FITC (ENF-FITC) (FITC: Fluorescein
Isothiocyanate is a derivative of fluorescein conjugated with an isothiocyanate functional group
(-N=C=S). fluorescein is a fluorophore, it has fluorescence activity at 460nm), at the

58. 58
concentration of 2,8mg/ml, and a stock solution of nanoformulated Enfuvirtide conjugated with
FITC (MYTS-ENF-FITC), at the concentration of 0,82 mg/ml, were both obtained by Dr Miriam
Colombo and Prof. Fabio Corsi from Sacco Hospital of Milan.
Efavirenz a non-nucleoside reverse transcriptase inhibitor, discovered by Merck, was used as a
control at a concentration known to be active against HIV-1 replication (1µM).
Enfuvirtide:
3.4 Experiments in vitro for the evaluation of antiviral activity of Enfuvirtide
C8166 cells were pretreated for 40 minutes with ENF 1: Fuzeon powder by Roche, ENF 2: ENF-
FITC-conjugated and ENF 3: Nanoformulated MYTS-ENF-FITC at different concentrations and
then infected with 10,000 pg/ml of HIV p24 for 2 hours at 37°C in an incubator. After 2 hours of
incubation, the cells were extensively washed two times with warm RPMI 1640 culture medium
without FBS to remove any excesses of the viral particles. Then cells were re-suspended in a

59. 59
complete medium (1ml) and cultured on 96-well plates for 7 days. 5 days after infection, cellular
cytopathic effect and syncytium formation were evaluated with (1) ENF (Fuzeon powder by
Roche), (2) ENF-FITC- and (3) Nanoformulated MYTS-ENF-FITC diluted in cell culture
medium.
To study the effectiveness of Fusion inhibitor different concentrations were used (0.1, 1, 10, 100
µM) for ENF 1: Fuzeon powder by Roche, ENF 2: ENF-FITC-conjugated and ENF 3:
Nanoformulated MYTS-ENF-FITC and the effect of replication was assessed at 7 days after the
infection of C8166 cells. In this experiment Efavirenz (1µM) was used as a control drug.
The inhibition of viral replication was evaluated by calculating both IC50 and IC90. IC50
represents the concentration of drug necessary to inhibit 50% of viral replication, while the IC90
is the concentration of drug adapted to inhibit 90% of the virus [70]
and is a direct measurement
of the potency of the drug itself.
The values are first obtained starting from the calculation of percentage of inhibition of each
drug at each given concentration tested by comparison with the untreated control (no drugs)
using the following formula,
% 𝑖𝑛𝑖𝑏𝑖𝑧 = 100 −
𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
𝑛𝑜𝑡 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
∗ 100
plotting the values of the percentage of inhibition, it is possible to calculate the IC50 and IC90
based on the use of the following formula:
𝐿𝑛𝐼𝐶50 = 𝐿𝑛𝐷 −
𝐴 − 50
𝐴 − 𝐵
∗ 𝑙𝑛
𝐷
𝐶
This method yields a sigmoidal dose response curve by relating the percentage of inhibition on
the y-axis with the concentrations of the drug on the x-axis in the semi-logarithmic scale.

60. 60
Figure: Dose-response curve useful to derive the formula to calculate the IC50. The point A
indicates the percentage of inhibition greater than 50%; B, the percentage less than 50%; C the
concentration of the drug in logarithmic scales to which is associated the effect B; D the
logarithmic concentration of the drug which is associated to inhibition of point A.
In the cell line C8166 [71]
, the IC50 and IC90 were obtained by evaluating the ability of the drug to
inhibit the syncytia effect of the virus and also by p24 measurement in the supernatants (see
paragraph 3.6).
3.5 Drug Toxicity
Drug toxicity was assessed in the absence of viral infection. Uninfected C8166 T cells were
treated in the presence of different concentrations of ENF 1: Fuzeon powder by Roche, ENF 2:
ENF-FITC-conjugated (up to 100 µM) and ENF 3: Nanoformulated MYTS-ENF-FITC (up to
20nM). Cell viability was visually assessed, and compared to untreated control.
3.6 Quantification of viral antigen HIV - p24
The GenscreenTM
HIV-1 Antigen Assay is an enzyme immunoassay (ELISA) for the detection in
vitro of p24 capsid of human immunodeficiency virus type 1 (HIV-1) in free serum, human
0
25
50
75
100
125
%
inhibition
Concentration
(nM)
A
B
C IC50 D

61. 61
plasma and the supernatant of cell culture. The test is based on the use of a solid phase prepared
with monoclonal anti-HIV-1 mouse, a first antibody biotin conjugate prepared with sheep anti p-
24 and a second conjugate prepared with Avidin linked to Horseradish peroxidase (HRP). The
samples and the controls are distributed to the appropriate wells within which is inserted above
the diluent supplied with the kit. From a stock containing 10,000 pg/ml of antigens, are made of
serial dilutions in PBS to be able to construct a standard curve based on known concentrations of
the antigens 200, 100, 50, 25 and 12.5 pg/ml. The plate thus prepared is left for 1h at 37 ° in a
dry-heat static incubator, and if they are present antigens of HIV-1, these will bind to the murine
monoclonal antibodies coated onto the wells and remain fixed during the whole time of the test.
Subsequently 5 washings are carried out in the plate with the washing solution (20X) diluted
1:20 composed of Tris buffer, NaCl at pH 7.4. The antibody biotinylated sheep anti-p24 is added
to the plate for 30 minutes at 37 ° C in a dry environment, that goes to fix the antigen-antibody
complexes on the bottom of the well. At this point 5 washings are performed as above and
adding the Avidin for 30 minutes at 37 ° C in a dry environment, that is to bind the antigen-
antibody complexes. The excess is eliminated through a further round of 5 washes.
For the detection of the reaction, the plate is incubated for 30 minutes at room temperature with a
chromogenic solution containing Tetramethyl Benzidine (TMB). The reaction is then stopped by
the addition of 1N sulfuric acid for about 5 minutes at room temperature. The optical density of
samples and controls is determined by spectrophotometry at a wavelength equal to 450nm-
620nm. The quantification of viral antigen present in the samples to be tested is made using a
reference standard curve prepared with known concentrations of p24 antigen supplied by Biorad.
The values of the antigen samples are calculated by interpolating the absorbance values obtained
with those of each concentration of the reference curve.

62. 62
CHAPTER 4
RESULTS
Results of BBB model in mice obtained by Dr Miriam Colombo and Prof. Fabio Corsi from
Sacco Hospital of Milan and the University of Milan demonstrated that Iron oxide nanoparticles
coated with an amphiphilic polymer increase Enfuvirtide translocation across the BBB in mice in
both in vitro and in vivo models. The mechanism involves the uptake of nanoconjugated-Enf in
the endothelial cells, the nanocomplex dissociation and the release of the peptide, which is
eventually excreted by the cells in the brain parenchyma. See figure below.

63. 63
4.1 Evaluation of antiviral activity of Enfuvirtide (ENF) and its conjugated forms with
FITC (ENF-FITC) and Nanoformulated (MYTS-ENF-FITC) in C8166 T cells
The antiviral activity of the fusion inhibitor was evaluated in HIV-1-infected C8166 T cells
treated with different doses of ENF, ENF-FITC and MYTS-ENF-FITC in in vitro colture
conditions. The inhibitory effect of ENF and ENF-FITC on viral replication was assessed and
compared by cytopathic effect based analysis of syncytium formation and quantification of HIV-
1 p24 gag Ag production in the supernatant cells at 5 and 7 days post infection, respectively.
Five days after HIV-1 infection, cells treated with either ENF or ENF-FITC showed a reduction
in the cytopathic effect in a dose-dependent manner compared to infected cells without any
treatments. (See figures below)
Figures notes:
The figure represents C8166 T-cells infected with 10, 000 pg/ml of HIV-1 using (pNL4-3; X4
virus strand) at 5 days post infection. Cells were treated ENF 1: Fuzeon powder by Roche, ENF
2: ENF-FITC-conjugated and ENF 3: Nanoformulated MYTS-ENF-FITC. Red arrows indicate
visible cytopathic effect (CPE).
Efavirenz (EFV) 1µM was used as control drug.
At 1µM Efavirenz completely inhibited HIV cytopathic effects, thereby, giving 100% protection
to the cells.

64. 64
Healthy cell, without viral infection and no drug, (A) showed no cytopathic effect (No
morphological change in the host cell, no formation of syncytia bodies, no cell lysis); differently,
in infected cells with no drugs, (B) we observed visible viral cytopathic effect.
Infected C8166 T cells were treated with ENF 1: Fuzeon powder by Roche, at different
concentration (0.1, 1, 10 and 100µM).

65. 65
In infected cells treated with ENF 1 at 0.1µM and 1µM, we observed some cytopathic effect (so
low protection from HIV infection), where as with ENF 1 at 10µM and 100µM we observed no
viral cytopathic effect, (so better protection from HIV infection).
Infected C8166 T cells were also treated with ENF 2: ENF-FITC-conjugated at different
concentration (0.1, 1, 10 and 100µM).
In infected cells treated with ENF 2 at 0.1µM and 1µM, we observed a reduced viral cytopathic
effect, with respect to ENF 1 at the same concentrations. (so better protection from HIV
infection). Differently, with ENF 2 at 10µM 100µM no viral cytopathic effect was visualized, (so
100% protection from HIV infection).

66. 66
Infected C8166 T cells were further treated with ENF 3: Nanoformulated MYTS-ENF-FITC at a
concentration of 20nM. At this concentration, we observed a high viral cytopathic effect due to
the low drug concentration.
By considering viral replication measured as HIV gag-p24 release in the cells supernatants, at
day 7-post infection, the C8166 cells in absence of drugs produced 979’465pg/ml of HIV gag
p24. By analyzing cells treated with different concentration of ENF and ENF-FITC a significant
reduction of p24 gag Ag production was observed: (See histogram below)
The histogram represent the total amount of HIV-1 gag p24 in the supernatant of C8166 T cells
infected with 10,000pg/ml of HIV-1 at7 days post infection. Cells were treated with ENF 1:
Fuzeon powder by Roche, ENF 2: ENF-FITC-conjugated and ENF 3: Nanoformulated MYTS-
ENF-FITC. Efavirenz (EFV) 1µM was used as control drug.

67. 67
At concentrations of 0.1µM of ENF 1 the viral production was 490’628pg/ml while at 0.1µM of
ENF 2 the viral production was 642’964pg/ml. Differently, at concentrations of 1µM of ENF 1
the viral production was 293’264pg/ml while at 1µM of ENF 2 the viral production was
364’246pg/ml. Finally, concentrations of 10µM and 100µM of both ENF 1 and ENF 2 the viral
production was 12,5pg/ml. With ENF 3: Nanoformulated MYTS-ENF-FITC at concentration of
20nM the viral replication was 1’345’000pg/ml.
By comparing viral replication results in treated sample and in the untreated sample, we
observed that: at 0.1µM ENF 1 showed 49.90% inhibition of the viral replication, while ENF 2
showed 34.35% inhibition. Differently, at 1µM ENF 1 showed 75.57% inhibition of the viral
replication, while ENF 2 showed 62.81% inhibition. At 10µM and 100µM both ENF 1 and ENF
2 showed 99.99% inhibition of the viral replication. Lastly ENF 3 showed 0% inhibition of the
viral replication.
The results of the p24 experiment were used to calculate the IC50 and IC90 of both drugs 7 days
post infection. Both ENF 1 and ENF 2 showed efficacy in inhibiting HIV 1 activity. ENF 1 had
IC50 and IC90 values of 0.1µM and 3.9µM respectively, while ENF 2 had IC50 and IC90 values of
0.3 µM and 5.4 µM respectively. See figure below.

68. 68
4.2 Evaluation of drug toxicity of Enfuvirtide and its conjugated forms with FITC (ENF-
FITC) and Nanoformulated (MYTS-ENF-FITC)
Drug toxicity was assessed in the absence of viral infection. Uninfected C8166 T cells were
treated in the presence of different concentrations of ENF 1 and ENF 2 (up to 100 µM, markedly
higher than the antiviral effective concentrations). Cell viability was visually assessed, and
compared to untreated control. No toxicity effect was found for all the compounds, tested at all
concentrations. See table and figures
Concentration drug (µM) Cell viability ENF 1, 2 Cell viability ENF 3
0 ++++ ++++
0,1 ++++ ++++
1 ++++ ++++
10 ++++ ++++
100 ++++ Not tested
Note: ++++ represent 100% viability (no toxicity)
ENF
1:
ENF
2:
0.1
0.3
3.9
5.4
IC50,
IC90
concentrations
C8166
IC50(uM)

69. 69
CHAPTER 5
DISCUSSION AND CONCLUSION
In this study, we aimed to test the antiviral activity of nanoformulated Enfuvirtide in the in-vitro
model of BBB. This result showed that nanoformulated MYTS-ENF-FITC is able to cross the
BBB model. The importance of the nanoformulated MYTS-ENF-FITC is to eradicate the HIV
virus from sanctuary sites in the CNS.
We tried the actual activity of the nanoformulated MYTS-ENF-FITC in human T-cell line
(C8166). Results obtained from the study of the cytopathic effect of the infected C8166 T –cells
treated with ENF 1, ENF 2 and ENF 3 demonstrated that in infected cells treated with ENF 1 and
ENF 2 we observed an increase in inhibition of the viral cytopathic effect and protection against
HIV in a dose dependent manner. The highest efficacy of Enfuvirtide against the cytopathic
effect of HIV-1 was observed with ENF 2 (FITC). The cells treated with ENF 3 did not show
protection from HIV infection due to the low drug concentration. Unfortunately a higher amount
of Enfuvirtide is needed to observe an actual effect in the BBB, so further investigation is in
progress.
Evaluation of the IC50 and IC90 of both ENF 1 and ENF 2 showed that ENF 1 had lower IC50
value of (0.1µM) with respect to ENF 2 (0.3µM). Results demonstrated that with ENF 2, to get
the same 50% inhibitory effect on the viral replication as ENF 1, a three-fold higher
concentration of ENF 2 is needed. The conjugated Enfuvirtide is required with respect to the
unconjugated Enfuvirtide. Differently, since ENF 3 (Nanoformulated MYTS-ENF-FITC) at
20nM showed no activity against HIV replication, its IC50 and IC90 values could not be
determined.

70. 70
From the analysis of viral replication measured as HIV-1 gag-p24, pg/ml release in the cells
supernatants treated with ENF 1 and ENF 2, both drugs showed a significant reduction of p24
antigen 7 days post treatment at different doses. Complete inhibition of the viral replication was
observed in both ENF 1 and ENF 2 at 10µM and 100µM showed inhibition. These results
demonstrated that at high concentrations, ENF 2 shows the same maximum efficacy as ENF 1.
Differently in the case of the nanoformulated MYTS-ENF-FITC (ENF 3), there was no
inhibition of the HIV replication (0%). This showed that the nanoformulated Enfuvirtide gave no
protection to the C8166 T-cells against the HIV virus.
Results from the toxicity showed 100% viability (no toxicity) in cells treated the nanoformulated
MYTS-ENF-FITC (ENF 3) showed no toxicity but its test was limited to its low concentration.
These results further proved that the conjugation of Enfuvirtide with FITC and Iron oxide
nanoparticles coated with a PMA shell is a positive modification to the Enfuvirtide scaffold.
Eradication of virus by sanctuary sites is a main goal in HIV management. The central nervous
system (CNS) is a classic model of sanctuary where viral replication occurs despite a complete
viral suppression in peripheral blood. In recent years, nanotechnologies have provided a great
promise in the eradication of HIV from the CNS. Dr Miriam Colombo and Prof. Fabio Corsi
from Sacco Hospital of Milan and the University of Milan demonstrated for the first time that the
structurally complex antiretroviral drug Enfuvirtide (Enf), which normally is unable to penetrate
the cerebrospinal fluid, is allowed to cross the blood brain barrier (BBB) in mice by conjugation
with a nanoconstruct [86]
.
The aim of this experiment was to demonstrate that Enfuvirtide (Enf) is able to penetrate the
cerebrospinal fluid into the blood brain barrier (BBB) in vitro in human C8166 T-cells by
conjugation with Iron oxide copolymeric nanoparticles. The ability of the nanoformulated ENF

71. 71
MYTS ENF-FITC to cross the BBB at 20nM was demonstrated; however it had no significant
efficacy against HIV replication due to its low drug concentration.
This project showed that to study the antiviral activity of the nanoformulated ENF MYTS ENF-
FITC against the HIV, a higher concentration of the drug is needed and probably a higher
concentration of the nanoparticle may also be required.
The major challenge with this experiment was the inability of the Iron oxide nanoparticles coated
with a PMA shell and functionalized on the surface with Enfuvirtide (MYTS) to contain more
than 20nM of Enfuvirtide. Future studies need to be done on the possibilities of optimizing the
Iron oxide nanoparticles to accommodate higher concentration of Enfuvirtide.
However past studies have shown that formulation of drugs with nanoparticles gives high
toxicity at therapeutic concentrations.
In 2009, Ochekpe N.A et al. cited that Nanotherapeutics might have unacceptable toxicity. The
very properties that make them useful may also lead to undesirable consequences. For example,
the larger surface area to volume ratio of certain nanomaterials may exaggerate their toxic effects
[30]
. They may be too large for renal clearance and if they cannot be degraded within the body,
they will accumulate, leading to nephrotoxicity [73, 74]
. Inorganic nanoparticles, in particular, may
not be easily degraded or metabolized, and once absorbed will remain in the body for years [72]
.
Another challenge of conjugating antivirals with nanoparticles is that there are technological
hurdles. Scaling up is challenging and expensive. Optimization at a laboratory scale is much
simpler than at an industrial or commercial level [74]
. The success of any nanopharmaceutical
depends on at least three criteria [75, 76, 77]
Firstly, the nanopharmaceutical should exert an
antiviral effect, secondly, it should have an acceptable toxicity profile and thirdly, it should be

72. 72
stable and be able to overcome biological barriers. The challenge is that optimizing one criterion
may be detrimental to the others, e.g., optimizing efficacy (by, for instance, including an
additional therapeutic agent into a multifunctional nanoparticle) may exacerbate toxicity
(because such an agent may be toxic) and decrease stability (because the more complex construct
is likely to be less stable). Extensive in vitro optimization experiments may be necessary to
achieve the ideal construct for in vivo evaluation.