Live bacterial vaccines use live attenuated bacteria to induce immune responses against target pathogens. There are two types - those that combat the disease-causing organism directly, and those that assist in combating other organisms by inducing immune responses against heterologous antigens carried by the bacteria. Currently licensed live bacterial vaccines include typhoid, cholera, and tuberculosis vaccines. Virulence is attenuated through genetic modifications like gene deletions or making the bacteria auxotrophic. Recombinant bacterial vectors can also be used to deliver heterologous protein or DNA antigens from pathogens. Effective vectors exploit bacteria that invade mucosal tissues to stimulate mucosal and systemic immune responses.
2. What is meant by live bacterial vaccines?
• Two types:
– Those that combat the disease causing organism itself
• An immune response induced against the bacteria itself
– Those that assist in combatting another disease causing
organism
• An immune response induced against a carried heterologous antigen
LIVE ATTENUATED
BACTERIAL VACCINES
RECOMBINANT BACTERIAL
VACCINE VECTORS
3. Live Attenuated Bacterial Vaccines
• Currently three of this type of vaccine exist:
Typhoid Vaccine – Ty21a
Cholera Vaccine - CVD 103-HgR
Tuberculosis - BCG
• Contains a live attenuated form of Salmonella typhi
Shigella – Proposed Vaccine
• Brand name is Dukoral
• Contains both a live attenuated version of Vibrio cholerae serotype O1
plus a recombinant cholera toxin B subunit (rCTB)
• Contains a live attenuated strain of the bovine strain of tuberculosis
– Mycobacterium bovis
MUST ENCOMPASS BOTH:
REACTOGENICITY
IMMUNOGENICTY
P
P
4. Virulence Attenuation
• Used to be achieved by laboratory passage but is now achieved by
recombinant DNA technology methods:
Gene Deletions
• Affect the way virulence genes are regulated
• Salmonella spp. deletions in phoP or phoQ genes.
Auxotrophs
• Require metabolites that are not present in host
tissues
• Gene deletions within the aro biosynthetic pathway
5. Recombinant Bacterial Vaccine Vectors
• Using live attenuated bacteria to carry heterologous antigens
– Either protein or DNA
Chromosomally Encoded Antigens
Plasmid Encoded Antigens
• Cassettes integrated into the chromosome
• Genetically stable
• Multiple antigen genes can be expressed
• Low copy number – only one per bacterial cell
• This may not be enough to elicit a suitable immune response
• Antigen genes encoded on plasmids
• Unstable – can be lost
• Amount of antigen expressed can be varied
• low copy and high copy plasmids
• Control of antigen expression via inducible promoters
dmsA dmsB dmsCIP
6. Getting Noticed
• Exploit invasive bacterial pathogens
• Vector type key to immune response – MHC Class I
Shigella spp. and Listeria monocytogenes
• Both are intracellular pathogens able to transverse directly
into the cytoplasmic region of the cell to deliver antigens to
MHC Class I pathway
• Auxotroph deletion asd – in vivo lysis due to low DAP
Salmonella spp.
• Delivery to MHC Class I is inefficient as antigens remain
localised to the membrane
• Exploiting the T3SS allows for efficient secretion of antigens
to antigen presenting cells
Immune responses can be achieved through efficient invasion of
mucosal cells of intestinal tract.
7. Positives, Negatives and
Future
• No injection – more compliance
• Easy, cheap to manufacture
• Potentially stable without refrigeration
• Multiple antigens in a single vector
• Can be destroyed with antibiotics
• Reversion to virulence
• Unwanted immune responses
• Environmental contamination
• Pathogens: HIV, those that attack GI, Respiratory and Genital tract
• Currently no licensed bacterial vaccine vectors – several currently in clinical trial
Positives
Negatives
Future
8. References
Carleton, H. A. (2010). Pathogenic bacteria as vaccine vectors: Teaching old bugs new tricks. Yale Journal of
Biology and Medicine, 83(4), 217-222.
Detmer, A. & Glenting, J. (2006). Live bacterial vaccines - a review and identification of potential hazards.
Microbial Cell Factories, 5.
Fiorentino, M., Levine, M. M., Sztein, M. B. & Fasano, A. (2014). Effect of wild-type Shigella species and
attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine
release. Plos One, 9(1).
Gunn, B. M., Wanda, S.-Y., Burshell, D., Wang, C. & Curtiss, R., III (2010). Construction of recombinant
attenuated Salmonella enterica serovar Typhimurium vaccine vector strains for safety in newborn and infant
mice. Clinical and Vaccine Immunology, 17(3), 354-362.
Jong, W. S., Daleke-Schermerhorn, M. H. & Luirink, J. (2014). An autotransporter display platform for the
development of multivalent recombinant bacterial vaccine vectors. Microbial Cell Factories, 13(162).
Kotton, C. N. & Hohmann, E. L. (2004). Enteric pathogens as vaccine vectors for foreign antigen delivery.
Infection and Immunity, 72(10), 5535-5547.
Orr, N., Galen, J. E. & Levine, M. M. (2001). Novel use of anaerobically induced promoter, dmsA, for controlled
expression of fragment C of tetanus toxin in live attenuated Salmonella enterica serovar Typhi strain CVD 908-
htrA. Vaccine, 19(13-14), 1694-1700.
Porwollik, S. (2010). Salmonella : from genome to function. Poole: Caister Academic Press.
Vecino, W. H., Morin, P. M., Agha, R., Jacobs, W. R. & Fennelly, G. J. (2002). Mucosal DNA vaccination with highly
attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for
T cells against HIV. Immunology Letters, 82(3), 197-204.
Wang, S., Kong, Q. & Curtiss, R., III (2013). New technologies in developing recombinant attenuated Salmonella
vaccine vectors. Microbial Pathogenesis, 58, 17-28.
Editor's Notes
What is meant by ‘live’ bacterial vaccines?
There are two types:
- live attenuated vaccines combat the disease causing organism itself and an immune response is induced against the bacteria itself
- recombinant bacterial vaccine vectors assist in combatting another disease causing organism and an immune response is induced against a carried heterologous antigen
Currently there are only three types of live attenuated vaccine available.
Tyhoid Vaccine – Ty21a which contains live attenuated Salmonella typhi
Cholera vaccine known as CVD 103-Hgr or by its brand name Dukoral - Contains both a live attenuated form of Vibrio cholerae serotype O1 and a recombinant cholera toxin B subunit known as rCTB.
BCG vaccine - contains a live attenuated strain of the bovine form of tuberculosis Mycobacterium bovis.
A vaccine for Shigella has also be proposed but as yet is not available.
To become a suitable candidate for vaccine the attenuated bacteria must encompass both low reactogenicity and high immunogenicity, if it has both of these it can be considered as a suitable bacteria vaccine.
For bacteria to be used for vaccine purposes they must first become attenuated to make them safe. Traditionally this was achieved by laboratory passage but now new techniques such as recombinant DNA technology are now used more frequently.
Deletion of either the phoP or phoQ genes – renders Salmonella extremely attenuated in vivo, as it cannot activate the two component regulatory pathway that helps the bacteria survive within the host.
Essential actions such as resistance to acidic pH, phagolysosome degradation and alteration of LPS to prevent detection by the host immunity, do not occur.
Examples include Salmonella Ty2 and Ty800 vaccines both of which have been effective at clinical trial.
Auxotrophs are those that require metabolites that are not present in host tissues.
Auxotrophs with gene deletions in the aro biosynthetic pathway cannot synthesise aromatic amino acids and as such only survive for limited periods of time in vivo.
This short survival time however is long enough for the desired immune response to be elicited.
Another use for attenuated bacteria vaccines such as Salmonella typhi is to use them as vectors to carry heterlogous antigens, such as DNA or proteins from other pathogens into the host.
Chromosomally encoded antigens have the benefit of being genetic stability and multiple antigens can be expressed, it does however provide only low copy number of the target antigen, as there can only be one per cell – this might not be enough to elicit a suitable immune response.
Plasmid can be either low or high copy number, but they can be unstable and the antigens can be lost. They do however have the added benefit of expression control via inducible promoters.
An example of this is the dmsA inducible promoter within Salmonella typhi – which is optimised for in vivo expression under anaerobic conditions.
Vaccine vectors exploit methods used by enteric pathogens which gain access to mucosal surfaces of intestine allowing antigens to then be delivered to immune cells.
Many viral and bacterial pathogens require the generation of cytotoxic CD8⁺ T-cells via the MHC Class I pathway for a successful protective immune response.
This kind of invasive methods is not a problem within pathogens such as Shigella spp. and Listeria monocytogenes which are both intracellular pathogens that are able to directly transverse into the cytoplasmic region where they will directly interact with the MHC Class I pathway.
Salmonella spp. however is inefficient at delivering antigens to the MHC class I pathway as it remains localised at the membrane of the host cell.
To overcome this the T3SS can be exploited to allow secretion of antigens into the host cell. For instance by fusion of an effector protein chaperone binding domain and secretion signal to the desired antigen.
This kind of antigen delivery has shown promise in mice whereby translocation and secretion domains of the outer membrane protein of Yersinia YopE were utilised for T3SS antigen delivery, by fusing these domains to two Listeria T cell antigens - listeriolysin O and p60. Mice vaccinated in this way showed good immunity through CD8 T-cell responses when challenged with L. monocytogenes.
Another example of delivery by this system is through fusion of H-2-restricted epitopes of the influenza virus with the Salmonella T3SS effector SopE. This is then able to interact directly with MHC Class I pathways.
Bacteria with auxotroph deletions such as the asd gene deletion which prevents biosynthesis of diaminopimelic acid (DAP) have shown to be effective at delivering antigens to the host cell - Shigella grown in absence of DAP will lyse in vivo – the host intracellular environment is low in DAP making this mutant perfect for effective delivery of vaccines in vivo. The asd mutation can also be used to ensure plasmid stability. When this gene is deleted from the chromosome it can instead be supplemented by being encoded on a plasmid which corrects the mutation.
Most of these vaccines can be taken orally dispensing with the need for needles and skilled medical professionals for administering of vaccines.
Easy and relatively cheap to manufacturer.
They are potentially stable without the need for refrigeration, allowing access to large communities of people even in isolated hard to reach places.
Multiple antigens can be expressed within one vector allowing for multiple pathogen vaccines which are potentially more economic. They can also be destroyed with antibiotics should an undesired immune response occur.
There is however a risk with using live vaccines, such as reversion to virulence and undesired immune responses, this is especially true in immune compromised people such as those suffering from HIV. There is also a concern that live vaccine cold contaminate the environment with genetically modified organisms and current opposition and lack of success at clinical trial make bring them to market difficult.
Many have performed well at clinical trial however and there are several pathogens that are proposed as being good candidates for this kind of vaccine delivery such as HIV.
Methods utilising live attenuated viral vaccines for HIV have been understandably negatively received, but research using Shigella and Salmonella as vectors for carrying sections of HIV DNA such as gp120 and gp140, have shown promise in mice that were able to produce good mucosal immune responses, such as TH2 cytokine production and TLR4 pathways following vaccination.
Bacteria resistant to many of the currently used antibiotics such as Streptococcus spp. are a major threat and some have even been isolated that are totally resistant. Bacterial vaccines used as vectors could allow for less use of antibiotics and could aid in the prevention of resistance arising. Currently there are no licensed bacterial vaccine vectors available but several have shown great promise at clinical trial. Ultimately getting the balance between reactogenicity and immunogenicity is key to any future vaccine success using these methods.
