Submitted by

Deepika kritaniya

Ph.D Biotechnology

(1st sem)

Human Immunodeficiency Virus (HIV) is a deadly andincurable illness impacting millions annually. Targeting the T-helper CD4+ cells of the immune system, HIV is a complexenveloped pseudo-diploid RNA virus. Of the nineteen proteinsencoded, one of the most readily produced and unique is thep24 inner capsid protein.

• The development of an HIV vaccine is a crucialcomponent required to bring the HIV/AIDS epidemic toan end.

• Vaccines can be either therapeutic or prophylactic.

• Therapeutic vaccines are designed to control/clear HIVfrom already infected individuals whereas,

• Prophylactic or preventive vaccines are designed toreduce the risk of infection in people who are notinfected with HIV and also to reduce the viral load setpoint in people infected with HIV.

Species of HIV

• There are currently two known species of HIV, classified as HIV-1and HIV-2.

• The virus is believed to be the result of at least 11 zoonoticinfections, arising from the transfer of simian immunodeficiencyvirus (SIV) in chimpanzees to humans.

• A study of genomic markers places the first SIV infection inchimpanzees sometime around the year 1492 and the migration tohumans by 1963. While HIV-2 is highly genetically similar to SIV,HIV-1 is both more virulent as well as more readily transferred.

• HIV-1 is classified into three groups, namely group M (main), O(other), and N (non-M/non-O), varying in slight geneticmodifications, mainly in the capsid proteins.

• Group M is further broken down into 9 subgroups, again with slightvariations. Further groups continue to arise as the result of geneticcombination of two or more varying strains.

Figure - HIV Virion Structure. Representation of HIVstructure. Gp120/gp41 complex on exterior, p17 matrixprotein, p24 capsid protein, and RNS bound with necessaryregulatory factors.

• The genes referred to as group specific antigen, polymerase,envelope, transactivators, and regulators.

• Group specific antigen (gag) is a single gene responsible forthe coding of four proteins, p6, p7, p17, and p24, all relatedto internal structural or core functions to the virus.

• Both p6 and p7 are nucleo capsids used to protect the RNAfrom degradation by way of nucleases, structured in a ratio ofone molecule RNA per hexemer of protein.

• p17 and p24 are both structural proteins, p24 being the maincomponent of the inner capsid, and p17 used as the anchor toattach the inner capsid to the outer envelope as well as theproteins found there in.

• Polymerase (pol) gene encodes three main proteinsresponsible for the reverse transcriptase, protease, andintegrases enzymes. Reverse transcriptase is the enzyme bywhich RNA is transcribed into DNA. In the case of Retrovirus,this is used in order to produce the material to insert the viralRNA genome into the host cell DNA genome.

• Envelope (env) encodes for the exterior viral proteins. Makinguse of a host cell enzyme Furin, the fusion protein gp160coded for my env, is cleaved into two smaller subunits, gp120and gp41.

• A gp41 trimer is directly attached to the exterior of the virionwith the trimer gp120 affixed onto the end of it. The role ofgp41 is that of mechanical cellular entry into CD4+ cells , asrecognized by the gp120 complex.

Transactivator• The purpose of a transactivator is to selectively

increase or decrease the rate of gene expression.• The three produced by HIV are transactivator of

transcription (tat), regulator of virion (rev), andviral protein-r (vpr).

• Tat works by enhancing the phosphorylation ofcellular factors, thereby increasing their expression.

• Rev is believed to assist with the exportation ofRNA from the cell nucleus to the ctytoplasm,allowing for the increased production of RNA.

• vpr may potentially work by accelerating theimport of viral precursor into the nucleus, to aid inRNA production.

Regulatory factors

• Three other miscellaneous regulatory factors are alsopresent within HIV, viral infectivity factor (vif), negativeregulatory factor (nef), and viral protein-u (vpu).

• Vif works to interrupt a main human antiviral protein,APOBEC, by targeting it for degradation by the bodies ownimmune system.

• Nef decreases the expression of MHC surface proteinsignals of the host cell, making it less likely to be markedfor degradation by the immune system, and therebyincreasing the chances of virion survival.

• Vpu is involved in one of the final step of virion production,the budding of the virion from the host cell.

Synopsis of completed Phase IIb/III efficacy trials of HIV vaccine.

TRIAL PRODUCT

CLADE

CANDIDATE

START

END COUNTRIES

NO.OF VOLUNTEERS

PHASE

IMMUNERESPONS

RESULT

VAX003

AIDSVAX

B/E Recombinant gp120 Protein

1988 2003 Canada, Netherland etc.

5,417 III T cell antibodies

No efficacy

VAX004

AIDSVAX

B/E Recombinant gp120 Protein

1988 2003 Thailand

2548 III T cell antibodies

No efficacy

STEP MRK-Ad5

B Ad5 gag/pol/nef

2005 2007 Australia, Braziletc.

3000 IIb T cell No efficacy

PHAMBILI

MRK-Ad5

B Ad5 gag/pol/nef

2005 2007 South Africa

801 IIb T cell No efficacy

RV144 ALVAC-HIV and AIDSVAX

B/E Recombinant gp120 Protein + Canary pox vector

2003 2009 Thiland

16402 III T cell antibodies

31.20%

HVTN 505

DNA and Ad5

A/B/C DNA Ad5 gag/pol/ nef

2009 2013 USA 2500 IIb T cell antibodies

No efficacy

• The RV144 trial was the only vaccine to show a modest effect andof the subsequent post hoc analysis done revealed a lot of usefulinformation. It is important to note that the RV144 trial resultindicates that developing a vaccine to prevent HIV infection ispossible.

• In addition to that, RV144 has offered a unique opportunity tocomprehensively search for immune correlates of risk of infection.This will provide a wealth of information to help reveal the immuneresponses necessary for protection against HIV infection.

• Genomic studies of HIV sequences from 110 participants from theRV144 trials also corroborated that antibodies are being targetedtowards V1V2 . A study also reported that the efficacy of thevaccine peaks around 12 months and gradually decreases indicatinga requirement for a booster dose to enhance the immune response.

Challenges Involved in the Design and Development of HIV Vaccine

• Massive diversity and variability of HIV presents a huge challenge to anefficacious vaccine design, as the vaccine needs to protect against aplethora of different strains . The vaccines studied to date are designedagainst one or two types of HIV clades.

• High level of difficulty in generating a vaccine that can activate CD4+ Tcells. Eliciting robust cellular and humoral immune response against abroad range of HIV subtypes.

• Lack of a human model showing complete recovery from HIV infectionand an appropriate animal model to predict the potency of an HIVvaccine. This makes it difficult to identify and induce immuneresponses required to cure HIV infection . This is supported by thefailure of VAX004 and VAX003 trials as the vaccine candidates weretested prior in non-human primate (NHPs).

• Lack of structural details of immunogens /antigens. Inability to makeantigens that mimic the conformation of the natural epitope.

• Non neutralizing antibodies interfering with protective response ofthe broadly neutralizing antibodies (bnAbs). Immune correlates ofthe RV144 trial have shown production of Non neutralizingantibodies.

• Person to person variability in T-cell and antibody responses inducedby the vaccine candidates . This was revealed from the study ofImmune correlates of the clinical trials.

• Designing an antigen binding to the B-Cell Receptor (BCR) with highaffinity. Accessibility of epitopes to antibodies .

• Study of the immune correlates of the vaccine trial indicates it isimportant to stimulate the production of both IgA and IgGantibodies.

• Clearance of latent virus. None of the trials have been shown toeffectively clear the latent viruses.

Designs and Strategies for HIV Vaccine

Attenuated and killed virus vaccines

These vaccines rely on direct mimicking of the viralinfection and have been tested in non-humanprimate (NHP). None of these approaches inducedneutralizing antibody responses and hence did notadvance to human trials. Setback of these vaccines isthe risk of reversal to pathogenic forms also posessafety concerns .

Recombinant protein subunit vaccines

• These are subunits of HIV surface proteins, example gp120and gp160, made using recombinant DNA technology.AIDSVAX used in the VAX003 and VAX004 trials was basedon recombinant gp120 (rgp120).

• The failure of the rgp120 based vaccine caused researchersto focus on developing soluble, recombinant Env trimers asantigens which resemble the functional HIV Env spike.

• Lately, a soluble trimer that closely mimics antigenicproperties of the natural Env trimer has been developedand shown to induce enhanced neutralizing antibodyresponses relative to monomers in guinea pigs .

• Discovery of broadly neutralizing antibodies (bnAbs) againstHIV-1 with high potency had significant consequences invaccine design. bnAbs namely PG9, PG16 and 10E8 showedneutralization breadth ranging from 80-98% .

• The bnAbs have been shown to bind to different sites likethe CD4 binding site (CD4bs), the first and second variableregions (V1/V2), the glycan-V3 site on gp120 and themembrane-proximal external region (MPER) of the gp41subunit. High potency bnAbs have been shown to providerobust protection against mucosal SHIV challenge in NHPs .

• Drawback of these vaccines is that they do not effectivelyinduce cellular immunity and also not mimic the naturalroute of infection.

DNA vaccines

• DNA vaccines contain a plasmid with a few HIV genes encodingproteins of interest that are inserted into the backbone. The vaccineis administered into the recipient and HIV proteins are expressed.

• The proteins are broken down into peptides and presented on thesurface of the cell for the immune system to respond. The DNAvaccines will deliver the genes without any immunity generatedagainst the vector itself.

• The most recent HVTN505 vaccine efficacy trial was a heterologousprime-boost strategy which evaluated a DNA vector expressing Gag,Pol, Nef, and Env as a prime and rAd5 vector expressed Gag, Pol,and Env as a booster.

• No DNA vaccines have yet been accepted for use in humans.Disadvantages of these vaccines is that they are limited to proteinimmunogen only, extended immunostimulation leads to chronicinflammation.

Live vectors

• Genes of interest are inserted into the genomes ofengineered virus to express the proteins inside the host.

• Viral vectors include non-disease causing viruses that arereplication defective in mammalian cells or viruses that wereengineered to be replication defective by deleting essentialgenes

• Genetic sequences that could encompass T-cell epitopes fromglobal HIV-1 strains have also been created. These sequencescalled mosaic sequences constructed for insertion into viralvectors .

• Adeno Associated virus (AAV), vectors with genesencoding for a bnAb against HIV-1 have been studied.This approach confers advantage of directly expressingbroadly neutralizing IgGs, thereby avoiding the difficultprocess of eliciting broadly neutralizing antibodies(bnAbs) by the immune cells.

• A recent study has shown that injection of AAV vectorswith inserts of genes encoding bnAbs into the musclesof humanized mice has conferred superior protectionagainst high doses of intravenous HIV challenges. Thedesign and development of viral vectors has been thestrongest of all other strategies used with somepromising results.

Peptide or protein vaccines

• This approach uses chemically synthesized proteinsubunits or peptides as vaccines to elicit cellular andhumoral immune responses.

• The disadvantage is these vaccines require adjuvantsto enhance the immunogenicity and currently alumis approved as the only adjuvant.

Virus like-particles (VLPs)

• VLPs are look-alikes of infectious virions containingempty shells of the HIV envelope protein; they lackviral genome and are therefore non-pathogenic.

• As VLPs resemble the virus, they are capable ofinducing high-level titers of bnAbs to protect againstHIV which exhibit multivalent structures.

• VLPs do not require adjuvant to elicit robust antibodyresponse as they are highly immunogenic themselves.

• Drawback of VLPs is that they stimulate strain-specificantibodies and induce an immune response againsthost cellular proteins, hence complicating the use ofVLPs as antigens.

Discussion

• For the last two decades or so conventional vaccinationmethodologies have so far been unsuccessful in elicitingstrong immune responses against HIV-1 and hence the field ofHIV vaccine research has focused on many alternative vaccinestrategies.

• It is imperative to note that a successful vaccine needs toelicit both B cell and T cell responses in order to be aneffective preventive vaccine.

• Integrated research endeavors in designing and developingantigens that elicit broadly neutralizing antibody responsesand protect against HIV infection, boosting up the T cellresponse to control viral replication and handling the problemof enormous HIV diversity are crucial elements in the pursuitof an HIV vaccine.

• Immune correlates of the modestly efficient RV144trial have identified the V1V2 region to be the target ofneutralizing antibodies and follow up studies areunderway to evaluate multiple prime-boost regimenswhich will generate more data and insights in to thedevelopment of a vaccine.

• Recent discovery of bnAbs has provided a new andexciting avenue for designing an efficient vaccine.

• Unique approaches like B-cell lineage vaccine and AAVvector vaccines to elicit bnAb responses are beingstudied.

References

• Nageswara Rao A (2014) The Pursuit of a HIV Vaccine – Trials, Challenges and Strategies. J AIDS Clin Res 5: 298. doi:10.4172/2155-6113.1000298.

• Design of Recombinant HIV Vaccine by Orthoreovirus 5’ Duplication,A Major Qualifying Project: Submitted to the Faculty of the WORCESTER POLYTEHCNIC INSTITUTE in partial fulfillment of the requirements for the Degree of Bachelor of Science in BiochemistryCameron W. Habib Thursday, April 29, 2010