HIV virus pre-targets CD4+ T cells

HIV virus pre-targets CD4+ T cells: Discuss the immunological consequence and what we should try to achieve with a vaccine

The human immunodeficiency virus (HIV) is a lentivirus and the etiologic agent of acquired immunodeficiency syndrome (AIDS). Its ability to target and invade CD4+ T cells has direct implications on the host immune system, beginning with the delivery of genetic information from the virus to the T cell, inducing a long period of latency and culminating in a progressive decrease in T cell numbers and loss of cell mediated immunity (CMI)1, 2.
The Immunological Consequence
The effect of HIV on the cells of the immune system has been well established and represents a series of crucial steps in the HIV lifecycle. Looking at HIV tropism, the virus demonstrates an affinity to bind to and replicate withinmacrophages and CD4+T cells, an attachment process mediated by the interaction of both the envelope glycoprotein (gp120) with the host CD4 molecules on the target cell1, 2. This process is assisted by the chemokine coreceptors CXCR4 located on the CD4+ T cells or an analogous CCR5 receptor on the macrophage which initiate structural alterations in the envelope protein 'env' (gp41), resulting in virion-host membrane fusion.

Implementation of the viral DNA into the host genome only occurs once attachment has taken place, indeed this represents a promising strategic target for vaccine generation1, 3. The net effect of the HIV infection can be seen in the serologic profile of figure 1; demonstrating immediate viral RNA detection during the acute phase of infection, whereby CD4+ T cells are being progressively destroyed until seroconversion occurs. Seroconversion sees the generation of the anti-HIV antibody by host B cells after propagation to the lymph nodes.

The immunological impact of this chronic phase is the continual generation and processive destruction of both CD4+ T cells and the HIV virions due to both a strong immune response, consisting of CD8+ CTL, antibody action and continual T cell invasion. In theory, the latent stage of infection does not see the virus lying dormant, as there is a persistent immune response toward the virus throughout the body. It is the imbalance of viral death to CD4+ T cell death which sees the subsequent decline in lymphocyte numbers. It is at this stage that an AIDS diagnosis can be made1.

A typical HIV-1 serologic profile demonstrating Tcell counts in response to viral load within the infected host. A diagnosisof AIDS is made corresponding to a T cell count of <200 cells/mm3(image adapted from Kindt TJ, Goldsby RA, Osborne BA. 'Kuby Immunology 6th Edition'. WH Freeman and Company. Chapter 20)

The consequence of the HIV virus preferentially targeting CD4+ T cells is the diminishing ability of the immune system to mount a delayed type hypersensitivity reaction. And as the infection progresses, the reduced ability for B cells to produce antibodies also becomes apparent. Having said this, the initial response to an HIV infection sees profound B cell dysfunction consisting of both hyperactivity and hyporesponsiveness1. A study into the B cell function of HIV patients demonstrated a diminution of IgM yet normal IgG production. B cells being generated with low CD21 expression, reduced IgM and non specific IgG may help to explain the poor humoral response to infection4. IgM is the primary antibody expressed on the B cells in response to an antigen; under normal conditions it binds antigen with very high avidity and is therefore an effective mediator of complement activation1, 3 . Impairment of IgM during HIV infection may be the first immune system defence failure, compromising the humoral response toward the virus.

It is not only the cells of the immune system which see significant modification in their expression. As the immune response is a tightly orchestrated, multifaceted scheme; signalling molecules or cytokines undergo a similar repression with a resultant knock-on effect on downstream cellular interactions1, 4. An HIV-1 infection sees significant dysregulation of cytokine production, secretion of Th(1) cytokines (IFN-gamma and IL-2) are reduced whilst the Th(2) cytokines (Il-4 and Il-10) and proinflammatory cytokines (IL-1/6/8) are increased in production4. The result of this irregular production contributes to the pathogenesis of HIV, again through the impairment of CMI. Cytokines are seen to aid the spread of HIV infection, through the expression of 'HIV cytokines', typically TNF-a/ and IL-1/6, which stimulate the induction of HIV replication in CD4+ cells.
Macrophages also see an upregulation in their surface CD40 and CD86 costimulatory molecules after infection, these molecules are implemented in the activation of T helper cells during antigen presentation. It should be stated that the combined effect of varying cytokine concentration elicits differing immune responses4, 5. For example, IL-2 given in high doses produces little effect of CD4+ cell numbers, however given for a short period of time at regular intervals, the effect on T cell stimulation is more profound. The underlying concepts of cytokine activation is currently being developed for 'cytokine therapy', drugs designed to boost the T cell humoral response to HIV infection6.
Vaccination Strategies
Currently, there are several well recognised antiretroviral strategies employed to help reduce the virulence of HIV, however it is finding the alternative treatment which will render HIV harmless which is proving to be the most challenging obstacle in the fight against AIDS1, 7.

HIV is unique in the fact that it is does not retain its antigenicity once incapacitated in its whole killed format. This therefore presents the initial hurdle. Coupling this with the fact that HIV is prone to genetic mutation upon replication, resulting in a lack of conserved antigenic regions on the virus, the design of new vaccines must achieve a high level of antigen specificity in order to induce a strong immune response8.

Recombinant Subunit Vaccines

The design and construction of a recombinant subunit vaccine (RSV) against HIV has been undertaken by several research teams9. Recombinant vaccines employ the idea of taking the most highly conserved regions of the HIV genome, those which demonstrate a considerable reduced ability to mutate. These regions can then be applied to vaccine vectors and injected into the subject. The ultimate goal is to develop memory T cells against the specific regions presented on the plasmid vector, culminating in a stronger, more potent immune response upon infection with the wild HIV-1 viral strain.
The generation of the novel HIV plasmid: 'HIVconsv' -was genetically designed to be suitable for a majority of vectors and likely to support high protein expression in vivo9. HIVconsv, a recombinant subunit vaccine contains only the most conserved sequences of HIV-1. It was generated synthetically containing humanised amino acid codons, an open reading frame proceeded by Kozak sequence (12 nucleotides which help maximise protein expression). 'Sven Letourneau, Eung-Junet et al' demonstrated that the vaccine was able to serve as a source of immunogenic epitopes within specific transgenic mouse strains. More promisingly, HIV-1 specific memory T cells were capable of recognizing HIVconsv derived peptides in 100% of HIV-1 infected patients - proving the fact that HIV-1consv specific responses are generated during wildtype HIV-1 infection.
The recombinant subunit vaccine's ability to associate with memory T cells is indeed a promising sign, and a step in the right direction to achieving a functional vaccine in vivo. However although the HIVconsv produced epitopes which were capable of being recognised, these epitopes were much less immunodominant than the more variable epitopes seen in the natural infection of HIV. In part, this may be due to the impact that these fragments may have on the intact epitopes, limiting their ability to be recognised by human Major Histocompatability Complexes (MHC)3, 8.
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The research put into other similar RSVs demonstrates the lengths one must go to in order to tackle the biggest obstruction associated with HIV; its level of diversity. To achieve a successful vaccine against any strain of HIV, it is clear that we should be looking for the most conserved regions of its genome in order to tailor the vaccine to a particular strain. Without a bespoke vaccine, HIV will continue to evade any vaccination efforts1,2,9.

Vectors

The type of vector used in a vaccine is critical to achieving an HIV induced specific immune response. The use of the Canarypox vector is very common place in designing effective vaccines, as Canarypox is able to enter human cells, yet it is unable to multiply and survive. Phase 3 trials of ALVAC and AIDSVAX demonstrated the effectiveness of selecting a suitable vector for HIV-1 strains (see fig.2)11. The high profile drugs ALVAC and AIDSVAX used Canarypox based prime-boost regimes which contained both gp120 and gp160 conserved residues capable of inducing both cellular and humoral responses across a very large patient group in Thailand.

Analysis of the ALVAC/AIDSVAX Canarypox vaccine demonstrating the reduction in probability of HIV infection following the administered vaccine. The data in the table below shows the number of infections witnessed in the trial population in both placebo and vaccine groups. (graph from Supachai Rerks-Ngarm, Punnee Pitisuttithum, Sorachai Nitayaphan. et.al. Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand, NEMJ,2009, 361.1056)

The underlying concept takes two known anti HIV drugs which worked synergistically; ALVAC priming and AIDSVAX boosting the immune response. The trial showed a significant, albeit modest reduction in the overall rate of HIV-1 infection, with a vaccine efficacy of 26.4% with a 95% confidence interval10. The vaccination was therefore able to induce an HIV specific response, a recorded increase in levels of IFN gamma when exposed to the 'env' or 'gag' protein of the wildtype viral strain.
Dendritic Cell Vaccines
Use of Dendritic cells (DCs) for an HIV vaccine represents a very plausible notion of DC based therapy11. Currently being employed as a novel and potent form of immunotherapy, it is utilised in the treatment of cancer and is currently undergoing phase 1 clinical trials for HIV12. Dendritic cells are bone marrow derived progenitors and a potent antigen presenting cell type, able to recognise, process and present foreign antigen to the humoral cells of the acquired immune system. DC therapy is able to exploit the natural pathways of antigen recognition, and may be able to help control HIV infection more efficiently by lowering the overall viral load once ART is removed.

By harnessing patient mononuclear cells, DC preparations can be generated in vitro. DCs from the patient must be differentiated from other monocytes within the medium (see fig.3). With a pure sample of DCs taken and cultured, the HIV antigenic proteins can be loaded onto the immature DCs and matured with recombinant CD40L which will ultimately result in maximal CD8+ T cell activation due to increased expression of IL-12 levels, eliciting a TH1 response1, 3, 11. The matured specific antigen presenting DC colony can then be administered to the patient where they will enter the blood stream and present the viral peptides to the maturing lymphocytes present in the lymphatic system, generating immunological memory against the virus.

A depiction of the vaccination process for dendritic cell therapy (ex vivo) showing the steps required for effective antigen loading onto immature DC cells and sequential maturation, DC, Dendritic cells; PBMC, Peripheral blood mononuclear cells; GM-CSF, granulocyte macrophage colony- stimulating factor
(image from: Rinaldo CR (2008) Dendritic cell-based human immunodeficiency virus vaccine (Review). JIntern Med; 265: 138-158.)

It is crucial to consider the choice of maturation agents and the specific viral antigens used which are likely to have an effect on the efficacy of any vaccine. Looking at the gp120 peptide as an example, and utilising it as the primary antigen for presentation on the dendritic cells, it may not be as effective at inducing an immune response as the alternative antigen gp160. In order to identify a prime antigen which will give the strongest response, experimentation and further study will need to be undertaken11.
Understanding the steps that need to be taken in order to put a vaccination model such as this one into practise is also important. Foremost, the type of dendritic cell that is to be harnessed will need to be selected. For example, either plasmacytoid, myeloid, langerhans or interstitial DCs; this is essential as different DCs express different cell surface molecules - DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DCSIGN) are expressed by langerhans cells and interstitial DCs, the ligands for these pattern recognition receptors have the potential to be powerful adjuvants in immunotherapy for HIV infection1, 11.

The methods for maturation of DC cultures will also require specific cytokines with the correct degree of costimulation. Cytokines are mediators of DC development, leading to effective CD4+ activation at the lymph nodes. A good requisite for generating anti-HIV T cell responses would be the production of IL-12, a heterodimeric cytokine which has a function in naive T cell differentiation. IL-12 is heavily implemented in the stimulation of growth and function not only T cells but the production of IFN-? and TNF-a from T and natural killer cells, enhancing overall cytotoxicity against HIV1, 5.
End Point Identification
Both immunological and virological end points are key to the development of an effective vaccination programme and not specific to just dendritic cell vaccines. Deciding on an effective end point allows for identification of vaccine efficacy, be it the stabilisation of the host polyfunctional T cells or a net increase in the overall magnitude of cytotoxic lymphocytes. By deciding on an end point, patient trials can be performed and the feasibility of the vaccine can be assessed.
The ALVAC/AIDSVAX trial initially established the 'presence of HIV infection' on the basis of repeat positive results on both an enzyme immunoassay and a western blot; from here they were able to set up an independent 'end point monitoring committee' whose members were able to verify the accuracy of the diagnoses made10. In turn this meant that researchers were ultimately able to identify whether the drugs administered were working in relation to their ideal outcome. In looking to tackle HIV, it is critical to know what must be achieved with a potential vaccine in order to reach an effective endpoint within a patient group.

Development of an effective HIV vaccine will see a paradigm shift in respect to the usage of antiretroviral therapies and will ultimately see a control to the rapid spread of the disease. Ideally, what we should plan to achieve is the production of a candidate vaccine that is not only effective in vitro but is able to hold up to phase 3 in vivo studies, generating a strong response to both arms of the immune system whilst producing as few side effects as possible.

References

[1] Kindt TJ, Goldsby RA, Osborne BA. 'Kuby Immunology 6th Edition'. WH Freeman and Company. Chapter 20; pp. 493-521
[2] Clapham P.Rand McKnight A. 'HIV-1 receptors and cell tropism'. British Medical Bulletin; 2001;58; pp.43-59
[3] Alfano M. andPoli G. 'The HIV lifecycle: Multiple Targets for Antiretroviral Agents'. Drug Design Reviews. January 2004 ; Volume 1,Number 1; pp. 83-92(10)
[4] Sahai BM, Dawood MR, Hammond GW. 'Diminished IgM but normal IgG production by purified B cells of HIV-infectedpatients in presence of accessory cells from HIV-seronegative donors'.National Conference on Human Retroviruses and Related Infections. 1993 Dec 12-16; 115.
[5] Wang J, Norcross MA. 'Effect of HIV-1 infection on cytokine and cell surface marker expression in human macrophages'. Conference on Retroviruses and Opportunistic Infections. 1997 Jan 22-26; 4: 148
[6] Levy Y et al. 'Safety, tolerability, pharmacokinetics, and efficacy of an interleukin-2 agonist among HIV-infected patients receiving highly active antiretroviral therapy'. Interferon Cytokine Res; 2008; 28(2); pp. 89-100
[7] Anthony S. FauciandGregory K. Folkers. 'Investing To Meet The Scientific Challenges Of HIV/AIDS'. Health Affairs; 2009; 28, no. 6; pp. 1629-1641
[8] Onafuwa-Nuga. A and Telesnitsky. A. 'The remarkable frequency of human immunodeficiency virus type 1genetic recombination'. Microbiol Mol Biol Rev.2009 Sep; 73(3); pp. 451-80
[9] Letourneau S, Im E-J, Mashishi T, Brereton C, Bridgeman A, et al. 'Design and Pre-Clinical Evaluation of a Universal HIV-1 Vaccine'. PLoS ONE; 2007; 2(10)
[10] Supachai Rerks-Ngarm, Punnee Pitisuttithum, Sorachai Nitayaphan, Jaranit Kaewkungwal, et.al. 'Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand'. New England Journal of Medicine; 2009;361
[11] Rinaldo CR. 'Dendritic cell-based human immunodeficiency virus vaccine' (Review). 2008; JIntern Med; 265: pp. 138-158.
[12] Sloan, Lois. 'Vaccination ofHIV-1 Infected Patients WithDendriticCells in Addition to Antiretroviral Treatment - (DALIA Trial)'.ClinicalTrials.gov identifier: NCT00796770

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