This document summarizes research into developing an anti-HIV vaccine using nucleoside-modified mRNA encoding envelope proteins. Key points: - Researchers developed mRNA vaccines optimized for protein expression by incorporating modified nucleosides, UTRs, caps, and tails to generate HIV envelope proteins. - Mice were primed with intradermal mRNA followed by an intramuscular protein boost to achieve strong T cell and B cell responses. - The mRNA vaccine induced high levels of IFN-γ, TNF-α, IL-2 in antigen-specific T cells and antibody titers, demonstrating the potential of nucleoside-modified mRNA vaccines for infectious diseases like HIV.

1. P41.25
Generating an Anti-HIV Vaccine Using Nucleoside-
modified mRNA Encoding Envelope
Norbert Pardi, Katalin Kariko, Michael Hogan, Hiromi Mur-
amatsu, James A. Hoxie, Drew Weissman
University of Pennsylvania, Department of Medicine, Phila-
delphia, PA, United States
Background: There has been great progress in understanding
the detailed molecular mechanisms of HIV-1 infection but no
effective vaccine has been developed to date. mRNA has
emerged as a very promising new therapeutic agent in recent
years and has already shown progress as an effective method for
generating potent vaccines.
Methods: To create a vaccine with maximal potency, in vitro
transcribed mRNAs were optimized for higher levels and ex-
tended translation by incorporation of selected UTRs, modified
nucleosides, 5¢ cap, 3¢ poly(A)-tail, and other modifications and
were HPLC-purified. To achieve both strong T cell and B cell
responses, heterologous mRNA prime - protein boost vacci-
nation regimens were used. For priming, naked mRNA en-
coding HIV envelope gp160 was administered intradermally
into mice. Cell surface Env was used to increase exposure of
neutralizing epitopes. Four weeks after the second mRNA
prime, Env protein was injected intramuscularly as a boost. As
nucleoside modified mRNA does not activate RNA sensors, to
increase the robustness of the immune response, a series of
adjuvant molecules and encoding mRNAs were co-injected
along with the antigen-encoding mRNA. Flow cytometry and
ELISA were used to evaluate T cell and B cell responses, re-
spectively.
Results: Elevated levels of IFN-v, TNF-a and IL-2 in antigen-
specific CD4+
and CD8+
T cells and high gp120 antibody titers
could be measured following two rounds of mRNA prime -
protein boost vaccination.
Conclusions: Our results demonstrate that antigen-encoding
nucleoside modified mRNA induces effective HIV-specific im-
mune responses and has great potential for vaccination against
infectious diseases.
P41.26
Comparative Neutralization Sensitivity of Indian
and South African HIV-1 Clade C Viruses to Plasma
Antibodies from Chronically Infected Indian Donors
Shilpa Patil1
, Sharda Gadhe2
, Swapnil Sonawane2
, Manish
Bansal1
, Suprit Deshpande1
, Tandile Hermanus3
, Lynn Morris3
,
K G Murugavel4
, Suniti Solomon4
, Seema Sahay2
, Ramesh
Paranjape2
, Bimal K. Chakrabarti1
, Jayanta Bhattacharya1
1
HIV Vaccine Translational Research Laboratory, THSTI-
IAVI HIV Vaccine Design Program, Gurgaon, India, 2
Na-
tional AIDS Research Institute, Pune, India, 3
National Institute
for Communicable Diseases, Johannesburg, South Africa,
4
Y.R. Gaitonde Centre for AIDS Research and Education,
Chennai, India
Background: HIV-1 clade C is the major subtype circulating in
India and South Africa. The present study was undertaken to
examine the extent of neutralization sensitivity of Indian and
South African (SA) HIV-1 clade C viruses to plasma antibodies
obtained from anti-retroviral naı¨ve chronically infected HIV-1
positive Indian patients.
Methods: Plasma samples from 150 anti retroviral naı¨ve donors
from India chronically infected with HIV-1 were assessed for
their degree of neutralization of 9 Indian and 10 South African
HIV-1 clade C envelopes using a TZM-bl reporter cell assay.
Reagents were shared between Indian and SA laboratories for
quality assessment. Antibody specificities were determined by
using TriMut core protein, mutant and chimeric viruses.
Results: 27/150 (18%) plasma samples were found to neutral-
ize > 50% of the panel viruses at 1:100 dilution. Amongst these,
seven were found (4.66%) displayed maximum breadth and
potency with median ID50s ranging from 300–750. The BCN
plasma antibodies were found to neutralize Indian (57%) viruses
slightly better than SA (43%) viruses. None of the potent broadly
cross neutralizing BCN plasmas was found to show neutralizing
antibody specificities targeting theCD4 binding site. However,
two of them showed N332 residuedependence in V3 loop in both
Indian and SA clade C Env backbones. While 3/7 BCN plasma
antibodies showed clade C MPER (HIV-2/HIV1 7312-C1C)
dependence, they did not show specificity to epitopes targeted by
known MPER directed monoclonal antibodies.
Conclusions: Our data suggest the presence of common epi-
topes in Indian and South African HIV-1 clade C envelopesas
most of the BCN plasma antibodies cross-neutralized pseudo-
typed viruses expressing clade C envelopes from both the
countries. Identification of epitopes common in both in Indian
and SA clade C envelopes will help define strategies to design
vaccine that would be effective in both countries.
P41.27
Cell-surface Display and Panning of HIV-1 Derived
Envelope Proteins
Tim-Henrik Bruun, Veronika Schmid, Alexander Kliche, Ralf
Wagner
University of Regensburg, Institute of Medical Microbiology
and Hygiene, Molecular Microbiology and Gene Therapy Unit,
Regensburg, Germany
Background: Available display systems allow the screening of
millions of candidate proteins and are the method of choice to
identify optimized antigen-antibody binding.
We established a mammalian cell-surface display to present
HIV-1 envelope derivatives in a natural, trimeric and membrane
bound environment. This allows us to generate affinity enhanced
envelope derivatives against broadly neutralizing antibodies
(bNAbs) in order to select potent Envelope (Env) based vaccine
candidates.
Methods: An HIV-1 derived lentiviral vector was developed to
infect HEK293T cells at a low multiplicity of infection (MOI),
in order to correlate phenotype and genotype. The vector was
designed and proven to express both GFP and Env in a constant
relationship, enabling indirect normalization for Env expression
by detecting GFP. After staining with an appropriate bNAb,
Env-displaying cells were selected for high affinity binding via
FACS-Sorting.
To adapt this system to the use of very large libraries, a refined
vector system was developed, allowing the stable genomic in-
tegration of both ENV and GFP at a distinct integration site. This
ensures that only one envelope variant is expressed per cell,
efficiently linking phenotype and genotype. Due to the stringent
linkage of ENV and GFP, GFP expression can again be used as a
means to normalize for Env expression in the FACS-Sorting
process.
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