No Effective HIV Vaccine Biology Essay

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The first recorded incidence of the human immunodeficiency virus (HIV) was in the Congo in 1959 (Zhu and Korber et al. 1998). It was believed to be transmitted to the West by a Haitian immigrant to the USA in 1960, long before the infamous Patient 0, Guten Dugas, was inappropriately identified in 1983 (van der Graaf and Diepersloot 1986). Although the clinical syndrome was identified comparatively early in the pandemic trajectory it took several years to identify the causative organism. A virus was suspected from the beginning but identifying it was a major hassle due to the amazing ability of the virus to mutate its protein coat therefore changing the structure of the epitopes in the binding region of the virus, which helps it to evade primary immune system recognition and also its entire genome. This means the virus can also re-emerge with current genes to create diversity (Asjö and Barin et al.1997). When this variation occurs in the regions which specifically encode for epitopes which are recognised by the cytotoxic T cells (CTL or CD+8 T cells) and the T helper lymphocytes (HTL or CD+4 Th cells), it gives a loop hole for the virus to 'hide' from these T cells and eventually proliferate into new strains of virus (Couillin and Connan et al. 1995).

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The mainstay of treatment thus far has been the promotion of the ABC method which stands for: "Abstinence, Be faithful, and use Condoms" (WHO 2005) for preventing the disease and the usage of anti-retro viral drugs for those that have contracted it. To date, the acquisition of the human immunodeficiency virus is effectively a death sentence as all who contract the virus will eventually die of its effects.

From recent statistics, it was reporting that in that one year there were about "five million new HIV infections, while nearly 3 million people died from AIDS, including approximately 500,000 children under the age of fifteen". (McCluskey and Alexander et al. 2005)

Taking a complete overview, it is clear that HIV not only fatally weakens the infected person's immune system but its effects are considerably more far-reaching in terms of the sociological impact, as it subverts the individuals' home life and status in the community. The effects of HIV are also felt in the work force as individuals take leave from work due to health which could either be permanently or for significantly long periods of time. Evidently this result in an increase in costs for the company involved this means hiring and training new staff for skills no longer being present in the workforce. It has directly increased health expenses and funeral costs. (Esparza and Bhamarapravati 2000)

Authorities such as Pilcher observe that the spread of HIV/AIDS has placed tremendous pressure on already insufficient health care systems on a world-wide basis. As the pandemic continues to spread, the world holds out for a vaccine as the best hope to stop its exponential growth. (Pilcher 2004)

In the initial years after the emergence of HIV/AIDS, a number of authorities confidently predicted the rapid development of an effective vaccine, but it was over two years before the pathogen was identified and characterised. (Heyward and MacQueen et al.1998). More than two decades later, no effective vaccine exists to provide effective human immunity against HIV infection.

This review considers the thrust of the medical and research community to find an effective vaccine by means of a thematic presentation of the research literature. It seems that it is unlikely that a conventional vaccine will be found because of the ability of the human immunodeficiency virus to mutate so readily but the vaccine candidates which will be highlighted in this review, both first and second generation, are targeted at vulnerable facets of the natural replication cycle of the virus and have achieved varying degrees of success.

However, before continuing any further, it is important to know exactly how HIV interacts within the body and the after effects.

3) Background

3.1) The structure of HIV

Figure 3.1a) "The proteins gp120 and gp41 together make up the spikes that project from HIV particles, while p17 forms the matrix and p24 forms the core."

The protein coat of the virus which is known as the viral envelope, consists of a lipid bilayer, that is formed from the membrane of a lymphocyte or the host cell whilst being released via exocytosis - this process is also known as budding (Zhang and Nguyen 2008). The glycoproteins (gp 120 and gp 41) or alternatively called exogenous antigens possess regions of binding called epitopes that enable a 'lock and key' formation with the receptor CD4 glycoprotein and co-receptors CCR5 or CXCR4) present on the CD4+ Th cells (a T helper lymphocyte or HTL) of the human immune system (Nielsen and Pedersen et al. 2005). The glycoproteins are typically made up of about 500 amino acids and "18 cysteine residues that form 9 covalent disulfide bridges. These disulfide bonds create a conserved series of simple and complex disulfide loops, 7 which in some cases define the boundaries of the five variable (V1-V5) and five conserved (C1-C5) domains of gp120" (Jobes and daoust et al. 2006) but the number of disulfide bonds remain the same and in positioning in virtually all the subtypes of HIV that are available. These glycoproteins will also end up on the surface of CD4+ Th cells, once infected from the virus.

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According to research digest (2005) these CD4+ Th cells are released in response to recognising foreign pathogens within the body i.e. viruses. CD4+ Th cells undergo no cytotoxic or phagocytic activity. Instead, cause activation of: macrophages (to engulf pathogens by phagocytosis), B cells (for production of antibodies) and more importantly in the case of HIV, CTL's formally known as CD8+ T cells (another type of lymphocyte possessing the CD8 glycoprotein as it's receptor) via the production of interleukins (signal molecules). CD8+ T cells are matured T cells with cytotoxic activity. These CD8+ T cells become specific towards the epitopes present on the HIV in order to 'kill' cells that are now infected by it. Initially this whole initiation results in a high positive activity towards HIV but with prolonged infection, the number of CD4+ T cells decline. CD4+ T cells are thought to decline in some of the following ways: the formation of gigantic synctium where by infected CD4+ T cells combine with non infected CD4+ T cells to give giant cell possesses 2-3 nuclei, infection occuring selectively, Memory Th cells made extinct (therefore if a past antigen enters the body, there will remain no memory of how the body will deal with this straight away), Normal CD4+ T cells made extinct in an abnormal process of some sort and routine responses from the immune system. However, recent research suggests that within infected individuals, a decline in CD4+ Th cells causes the activation of "cell death" (alternatively known as apoptosis) rather than generating new numbers of these cells. (Gougeon and Montagnier 1993)

"What turns on the cell death program? Apoptosis might be triggered indirectly (left) or directly (right) in patients with AIDS". (Gougeon and Montagnier 1993)

Generally for infected individuals, a decline in the number of CD4+ Th cells means the rate at which CD4+ Th cells regenerate too also declines. However, the numbers of CD8+ T cells in the meanwhile continue to increase and some also differentiate. The differentiated CD8+ T cells unfortunately do not have the function of targeting and killing HIV and consequently lose the ability to multiply in numbers also. This means all the pressure is put on current active CD8+ T cells. For a short period of time, these cells perform to their peak but then they eventually retire and become anergic. All this unfortunately leads to reduced immunity, therefore making one more susceptible to other diseases.

3.2) The HIV life cycle (campbell and hope 2008).

3.2.1) Reverse Transcription followed by integration

Upon the virus fusing with a CD4+ Th cell via endocytosis, the transformation of viral RNA takes place to form viral DNA by reverse transcriptase. Integrase enzyme allows viral DNA to be incorporated into the human DNA which later needs to be activated.

3.2.2) Transcription followed by translation

The virus is then activated at any period of time by converting it to messenger RNA with use of the enzymes present from the host cell. Messenger RNA is then used to form viral proteins such as glycoproteins which migrate towards the host cell membrane and bud off to release new viruses. The enzyme protease is then used in producing mature viral cores that can then repeat the above cycle again and affect other cells of the body

Figure 3.2a) (Nielsen and Pedersen et al. 2005) A cartoon showing the various stages of the HIV life cycle: 1. Virus-receptor interactions, 2. Virus entry, 3. Reverse transcription, 4. Proviral integration, 5. Transcription, 6. Splicing into human DNA followed by nuclear export, 7-9. Translation, 10. Assembly, release and maturation

4) Antiretroviral treatments - from past to present.

Antiretroviral medication is the term used for medications used in suppressing the HIV from inflicting any more damage on the human immune system within infected individuals. When an amalgamation of two drugs or three or more drugs are used, these are referred to as a combination therapy and Highly Active Antiretroviral Therapy (HAART) respectively. (Kis and Robillard 2009). Combining drugs may have the following effects: "additive, the activity of the drugs in the combination is equal to the sum of the activity of each drug when used alone; synergistic-the activity of the drugs in the combination is greater than the sum of the activity of each drug when used alone; and antagonistic-the activity of the drugs in the combination is less than the sum of the activity of each drug when used individually" (look at combination therapy). The various drugs currently available will discussed briefly followed by a discussion of any limitations these may have.

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4.1) Nucleoside or Nucleotide reverse transcriptase inhibitors

Nucleoside reverse transcriptase inhibitors (NRTI's) were the first drugs to be synthesised these were namely: "AZT (zidovudine), ddI (didanosine) and ddC (zalcitabine) with respective dates of US approval of 1987, 1991 and 1992" (Martin and Hitchcock et al,. 2009). Others also later followed: "stavudine (d4T), lamivudine (3TC), abacavir (ABC), tenofovir disoproxil fumarate [TDF; prodrug for the oral delivery of the nucleotide analog tenofovir (TFV)] and, most recently in 2003, emtricitabine (FTC)". NRTI's or NSRTI's are administered as prodrugs and undergo a series of phosphorylation steps by the enzymes within a CD4+ Th cell: kinase and phosphotransferases to form deoxynucleoside triphosphate (dNTP) type substrates (Cihlar and Ray 2009). What differentiates these substrates from natural deoxynucleotides is the removal of a hydroxyl group in the 3 position of the deoxyribose part. Without the hydroxyl group, formation of the phosphodiester bond in the 5' to 3' direction of DNA sythesis is not possible. Therefore as the name suggests, they inhibit enzymatic sites on the HIV reverse transcriptase which disrupt the synthesis of HIV replication.

Figure 4.1a) some structures of NRTI's

A review by Dickinson et al. (2010) show possible drug-drug interactions and drug toxicity, whilst Hawkins (2010) lists all the possible adverse side effects. Side effects will be discussed in more detail later.

4.2) Protease Inhibitors (PI's)

PI's were the next generation of HIV medication to be synthesised after the genomic Pol region HIV was discovered. Here lie codons which code synthesis for: HIV protease as well as reverse transcriptase and integrase enzymes. Initially, the enzymes are formed as precursor polyproteins. These are then cleaved by already present HIV protease to produce their prospective matured enzymes which will be used in formation of a new virus. Protease inhibitors interrupt the cleavage process therefore disrupting maturation.

There are currently ten approved drugs by the FDA: some of which are Amprenavir and tipranavir. (http://www.fda.gov/ForConsumers/byAudience/ForPatientAdvocates/HIVandAIDSActivities/ucm118915.htm)

4.3) Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

NNRTIs are similar in respect to NRTI's and NtRTI's in that they upon reverse transcriptase, but their mode of inhibition is completely different. They bind to a different active site with respect to NRTI's and NtRTI's and so are not included into the synthesis viral DNA. NNRTIs make reverse transcriptase immobile and so it is unable to catalyze the form phosphodiester bonds in the 5' to 3' direction.

4.4) Fusion/Entry Inhibitors

The majority of drugs discussed so far have been directed at targeting HIV when inside a CD4+ Th cell, but fusion/entry inhibitors aim for virus entry instead. These intercept how the virus binds, fuses and enters a CD4+ Th cell. Preventing the virus into a cell altogether means the immune system will hopefully stay intact.

4.5) Integrase inhibitors (II's)

Raltegravir and Elvitegravir are the main inhibitors available currently. The binding site of the enzyme contains bivalent metal cation cofactors that allow covalent bonding to human DNA. However, the integrase inhibitors bind to these instead therefore obstructing integration of proviral DNA into human DNA.

Anti-retroviral drugs - the pros and cons

Initially with NRTI's, monotherapy drug treatment started with some degree of success; later followed by combination therapy. AZT with ddI or ddC, had additive or synergistic effects but the suppression of HIV with these drugs was only temporary and needed to be administered to patients quite early on in their HIV prognosis i.e. when their CD4+ Th cell count was of at least 150 cells/mm3. With any combination of these drugs, as infection increased, drug resistance too also increased. A combination of ddl and ddC proved to be particularly fatal in causing multi-drug resistance among HIV (Jablonowski 1995).

PI's can alter the inward functioning of cells and cell lifetime but as a consequence they're considered to be useful as anti- tumour agents via inhibiting the multiplication process of cancer cells in certain clinical trials (Danaher and Wang et. al 2010). Second generation PI's in general showed better bioavailability, half-life and lower toxicity. A study by (Cameron and Heath-Chiozzi et al. 1998) showed that within a majority patients who initially tried at least one type of NRTI's and had a CD4+ Th cell count of 100/µL or less, enabled HIV-RNA to be under 200 copies/mL, which is always considerably more. Drug resistance to PI's occur due to mutations to: the gag protein and to the overall binding site of protease enzyme by increasing in size (Wensing and Maarseveen et. al 2010).

Etravirine a NNRTI, is a relatively new drug on the market that is used in combination with other anti-retroviral drugs (Bahal and Romansky et. al 2003). In a study by Bahal et. al, Etravirine showed a low bioavailability of 9 mg/mL in water and a burning aftertaste that incurred shortly after swallowing the drug; a taste that deterred many patients from consuming it. Therefore a different formulation of the drug needed to be synthesised in order to tackle these problems. Medium chain triglycerides (MCT) were used to increase solubility to 150 mg/mL in( MCT) and the use of sucrose and xylitol changed to give a taste of that similar to that of icing sugar. However another study by showed a Etravine also works against some mutants of HIV-1 that are resistant to other NNRTI's such as Nevirapine and Efavirenz (http://www.fda.gov/ForConsumers/ByAudience/ForPatientAdvocates/HIVandAIDSActivities/ucm124019.htm).

Fusion/entry inhibitors are relatively new to the market, and are only used if other methods of combination therapy have failed. However, a study by Hartley and Gaertner et al shows quite the opposite and how vital the CCR5 co receptor is in the mechanism of HIV transmission and how other evidence strongly suggest that its inactivation would not generate adverse side effects. (Dean and Carrington et al. 2006)

Other examples of entry inhibitors include Enfuvirtide. This drug is likely to suppress HIV in the short term, but will lead to drug resistance relatively quickly. However it has been shown to be quite potent against HIV that is resistant towards PI's and RTI's (redshaw and westby 2001).

As mentioned earlier, the principle of mutation to the enzymatic binding site can be applicable to most drug resistance that occurs with the majority of anti-retroviral drugs such as Raltegravir. The CD4+ cell count for Raltegravir in relation to Efavirenz is 189 cells/mm3:163 cells/mm3.

Clearly with many of the anti-retroviral drugs the problem is mainly drug resistance. As a result anti-retroviral treatment cannot be used alone to fully suppress HIV. Therefore the only way forward would have to be the generation of a vaccine which would prevent anyone at risk to the virus from contracting it.

(I will add more to this! Section in drugs by drawing more drug structures etc)

6) Detection of AIDS virus using ELISA techniques

Arguably one of the most useful biochemical tools in the determination of any vaccine is the ELISA Assay (Enzyme-linked immunosorbent assay). This is a type of assay (measurement of activity) used to show the presence of an antibody or an antigen is a sample. The majority of the studies presented utilise ELISA or a close variant, and it is worth therefore considering the theory of the process.

The mechanism behind the assay is that a sample of antigen is fixed to a non-reactive surface, most usually a polystyrene sheet, and is then washed with an antibody specific for the antigen and then allowed to bind. After a period for reaction, the substrate plate is gently washed to remove unbounded excess of the antibody. The antibody is linked to a signal enzyme which allows a quantitative measure of the fixed antibody. Most commonly, fluorescence is the quantity measured and this can be indirectly related to the amount of antigen present in the original sample. (Christiansen and Jessen et al. 2006)

More recent variants of ELISA include:

Real-time PCR - This technique enables the amplification of viral DNA by the use of primers that are approximately 20 nucleotides in size. These are complimentary to a sequence of bases (i.e. 100 - 600 bases in size) present on the DNA. DNA polymerase lengthens the primers so that full complimentary strands of DNA are made. The process is repeated several times and hence gives many copies. (Antinori and Calattini et al. 2007)

Fluorogenic and electrochemiluminescent techniques which allow very high degrees of sensitivity. These assays are not always "enzyme - linked" and are therefore not technically ELISAs, but the underlying principles are the same. (Pandori and Hackett et al. 2009)

5) HIV/AIDS vaccine. Goals and timeline

Championing the effects of HIV started with the "ABC" effect as mentioned, but this alone cannot work. Therefore a vaccine would be advantageous and to work alongside current anti-retroviral drugs.

The benefits of a human immunodeficiency virus vaccine may not be immediately obvious. Catanzaro and Graham offer an authoritative overview of the probable attributes of a human immunodeficiency virus vaccine by commenting on the fact that classical vaccines provoke an immune response to keep infection at bay and stop it from progressing any further or to stop contracting the virus altogether. For now the evidence suggests that the most likely first candidates for a human immunodeficiency virus vaccine will provide only partial protection. (Blankson and Persaud et al. 2002).

induce immunity to prevent infection or to keep an infection from persisting and causing disease

What is interesting also, present evidence suggests that the initial vaccines developed, can only provide immunity to some cells. But this does show that there is an ability to decrease the rate of infection, until a good immune response kicks in to clear the infected cells. This would suggest that the progression of the disease processes would be slowed thereby reducing and delaying the need for Anti retroviral treatment. Such a vaccine could also be expected to augment conventional therapies by requiring that the virus has to mutate both the immunologically sensitive sequences as well as the active sites of Anti-retroviral drugs in order to avoid removal (Catanzaro and Graham 2009).

There is also the corollary point, as Longini et al. have observed, that an additional goal of a HIV vaccine would be to create a degree of immunity that allows a reduced ability for the infected person to transmit the human immunodeficiency virus to others by decreasing the person's viral load. It clearly follows that realising this goal would benefit the general population at large by slowing the spread of the pandemic over time. (Longini and Datta et al. 2006)

Kinloch-de Loes et al. suggest part of the reasoning for developing a vaccine is that many HIV infections occur via direct contact with an individual with a high viral load during the primary stages of the disease. The infected person may be unaware of their HIV positive status and so might not have started to obtain treatment for it or to curb their sexual activity (Kinloch-de Loes and Autran 2002)

7) Major problems in vaccine development

As mentioned, the virus can hide from immune responses quite cleverly. It has proved virtually impossible to make a vaccine containing antigens that can invoke an immune response of antibodies that can tackle all sorts of viral strains. Therefore virtually all who become infected with HIV eventually die from AIDS. (Glynn and Biraro 2009)

One has to acknowledge both the additional social and ethical barriers to vaccine development, but these are mainly tangential to the main thrust of this review and therefore will not be discussed further.

8) The ability of an agent to block human immunodeficiency virus entry into cells.

The term "agent" can be applicable to any drug, vaccine, chemical or protein which could stop the virus entering the cell. An early study into the difficulties of producing a vaccine was offered by LaCasse et al. and is considered in a quasi-historical context to illustrate the early difficulties and concepts and to offer contrast to the later studies. (LaCasse and Follis et al. 1999)

It has to be noted that this study was conceived in the aftermath of studies which had showed that early rgp120 formulated vaccines, which were effectively weakened forms of gp120 exogenous antigens from people already infected with HIV/AIDS, initiated an immune response that dampened the effects of HIV. Although not completely, with the results typically in the region of 30 to 50% neutralisation. (viz. Mascola T R 1996)

As already discussed, HIV is engaged in a sequence of interactions that consequently lead to the fusion of viral and CD 4 memembranes. On this understanding, LaCasse et al. set out to explore human immunodeficiency virus immunogens that explicitly incorporate these functional intermediate structures with a view to arresting the process before cell infection could be accomplished.

HIV-1 defeats host-mediated resistance by CEM15

http://www.biocarta.com/pathfiles/h_vifPathway.asp

Synthesis of the vaccine: gp120

This was conducted by culturing CD4 cells with their respective and CCR5 co-receptors with engineered infected CD4 cells that possess an envelope protein. The process of binding and fusion was followed by inducing formaldehyde cross-linking before many infected HIV cells congregated with non infected CD4 cells to form large cells (synctium). To prevent this, the culture was placed in 0.2% methanal for 5 hours. The fusion-competent immunogen (FCI) was now synthesised.

FCI was then injected into transgenic mice that also have CD4 and hu CCR5 co-receptor. In response to the FCI, antibodies formed and were found to neutralise human HIV; thus making an excellent serum. A mechanism described in a note by LaCasse et al : "involves the serum being adsorbed sequentially to protein A Sepharose (Sigma) and protein G agarose (Sigma) at 4°C. The adsorption of antibody was confirmed by gp120 ELISA. These solid supports were then combined and antibodies were eluted with 100 mM glycine, pH 2.5. The eluate can then be neutralized and dialyzed by centrifugal ultra filtration".

It was hoped that, by targeting the mechanisms that allowed HIV and cells to unite, that this might circumvent the difficulties imposed by the changing antigenicity of the human immunodeficiency virus envelope. Sadly subsequent clinical testing did not support this optimism. In fact, many of the research conducted on HIV envelope proteins did not progress further from phase one, as the antibodies synthesised only neutralised HIV cultured in a lab and not against any wild type HIV strains.

The recognition of an ability of an agent to block human immunodeficiency virus entry into cells, although not strictly a "vaccine" in the true sense of the word, has caused a number of research teams to begin working on an agent which could block the ability of human immunodeficiency virus to enter the cells. In this respect one can consider the review paper by Hartley et al. The paper begins with an overview of the (then) current state of therapeutics and the reason why many antiretroviral therapies appear to become less effective with time as they are directed against one particular element of the viral entity, which then changes in morphology or function. (Hartley and Gaertner et al. 2004)

Hartley and Gaertner et al shows how vital the CCR5 co receptor is in the mechanism of HIV transmission and how other evidence strongly suggest that its inactivation would not generate adverse side effects. (Dean and Carrington et al. 2006)

The paper begins with an overview of the (then) current state of therapeutics and the reason why many antiretroviral therapies appear to become less effective with time as they are directed against one particular element of the viral entity, which then changes in morphology or function. (Hartley and Gaertner et al. 2004)

RANTES is the main natural ligand of the CCR5 receptor and is found to prevent R5-tropic HIV strains entering the CD4+ Th cell.

Hartley and Gaertner et al believe in that RANTES binds to CCR5 with high affinity and in the correct orientation leading to stable host guest formation. RANTES also possesses the Nitrogen - terminal region in which the receptor is activated. They thought that if similar analogues to RANTES could be synthesised thus exploiting the attributes of RANTES, then an effective blocking agent could be made. 37 analogues of RANTES were made that contained various substituents' that differed from the original. Screening activity of these led to the emergence of PSC-RANTES {N-nonanoyl, des-Ser1[l-thioproline2, l-cyclohexylglycine3]-RANTES(2-68)}, that was 50 times more active than AOP-RANTES, the first synthetic CCR5 cytokine to be trialled. (Hartley and Gaertner et al. 2004)

The following schematic shows how to make elongated AOP- RANTES. However, the same principles can be applied to making PSC RANTES.

The Pentyl oxy amino acetaldehyde or AOP constituent of the polymer supported RANTES, couples to a carboxylic acid (COOH) linked to a hydrophobic chain via the terminal nitrogen in aqueous solution at PH7; effectively the COOH undergoes a boc protection (1 combines with 2). Steps (i) basically removes the solid support and following step (ii) a peptide formation occurs (ligation) with RANTES which is purified to give folding and di-sulfide formation. (Wilken and Hoover et al. 1999)

Figure (Wilken and Hoover et al. 1999)

In vivo, the mice were injected with PSC-RANTES followed by R5-tropic HIV strains. While another set of mice were injected with just the R5-tropic HIV strains. The results were highly significant in that the viral RNA reached less than 200 copies per ml, which could not be detected for an entire month. Whereas all the infected mice had viral RNA greater than 10,000 copies per ml, 2 weeks in to the experiment alone.

In light of this study, the authors believe that there is now a promising future into looking at other small proteins.

9) Epitope modulation in regard to vaccine production.

The chemistry of the immunological response in relation to a vaccine.

A further step on the road to human immunodeficiency virus vaccine development can be seen in the Okazaki study where the authors set out to produce an enhanced CD4 human immunodeficiency virus epitope. The authors point out that the CD4+ T help cells appears to play a critical role in maintaining CD8+ Th function in viral infection (viz. Hasenkrug and Brooks et al. 1998)

It is a characteristic of human immunodeficiency virus infection that there is a quantitative decline in the number of CD4+ lymphocytes and a qualitative impairment of CD4+ T cell function which inevitably lead to the development of AIDS. The rationale for this work revolves around the fact that the HIV-specific CD4+ T cell response can be recovered after initiation of highly active antiretroviral therapy. It appears that stronger CD4+ Th cells are required for the maintenance of CD8+ Th and control of how the virus enters the bloodstream (viraemia).

In the context of vaccine production, it is thought that the virus might be controlled by a vaccine incorporating improved CD4+Th cell epitopes of HIV where the Amino acid sequence has been changed for a better immune response from the CD4+ Th cells . This was thought to hopefully generate large numbers of specific CD8+ Th cells, together with similarly enhanced CD8+ Th receptors, a concept first put forward by Berzofsky et al. (Berzofsky and Ahlers et al. 2001)

The paper itself describes the investigation where the T1 Ag, which is a 16-mer peptide (KQIINMWQEVGKAMYA) epitope for the CD4+Th cell. This was the first helper epitope characterised from the HIV envelope protein (Cease and Margalit et al. 1987) and the same authors showed that immunisation with it, is recognized by T helper lymphocyte cells, with people that are infected with HIV. Ahlers et al. subsequently showed that the activity of this epitope could be significantly increased when it was chemically manipulated to replace 436E (glutamic acid) in the T1 epitope is with A (alanine), at least in the mouse model (Ahlers and Belyakov et al. 2001). The rationale for the Okazaki study was to boost the activity of the CD4+ Th cells in humans by exposure to the stimulus of an epitope-enhanced T1 peptide. So in both studies, the authors thought that if the epitope was isolated and injected and then later enhanced by Okazaki, it would trigger an immune response without having to inject the whole of the virus.

This concept was expanded further by the McKinney et al. study which specifically considered vaccine strategies that had been developed to address HIV-1 variation and which made use of ancestral genes or consensus genes based their different subtypes and using epitopes that are in the preserved and variable in regions on gp120 (McKinney and Skvoretz 2004)

The HIV genome has regions which are believed to be preserved and to remain like this till the virus eventually dies. This is because mutations would perturb how DNA replication and synthesis of proteins e.t.c. would take place therefore effecting HIV survival. The authors point out that analysis of HIV-1 sequences demonstrates that epitopes that code for CD8+ T cells mainly reside in these preserved regions on gp120; whereas regions that are lacking of epitopes that code for CD8+ T cell are in the variable domains of gp120 (citing Yusim and Kesmir et al. 2002). They also believe that CD8+ Th cell recognition occurs with all the different HIV-1 subtypes that have been discovered during natural infection and vaccine investigations (Fukada and Tomiyama et al. 2002). The authors argued that: "it is the fact that recognition of conserved epitopes which have been derived from diverse HIV-1 subtypes by CD8+ Th cells that supports the development of epitope-based vaccines". But on the other hand of the argument, more than 40% of the isolated CD8+ T epitopes from these preserved regions could not code for the most common strains HIV. (McKinney and Skvoretz 2004)

They synthesised epitopes containing oligonucleotides in a polymerase chain reaction by an overlapping method which is further described in the paper by (Ishioka and Fikes et al. 1999)

Mckinney et al. showed some of the modified epitopes from these preserved regions interact with T helper cell receptors incorrectly i.e. no immune response is initiated to cause the release of B cells to engulf or activate of CD8+ T cells e.t.c. Therefore at times, these epitopes effectively acted like antagonists (Klenerman and Rowland Jones et al. 2004). These antagonistic epitopes are not the only synthetic ones, but some occur naturally. Antagonism of the CD4+ Th cell population has been found in a previous vaccine study already discussed by Hartley and Gaertner et al. 2004 and this is quite worrying as one patient was found to be infected with HIV after injection of the vaccine. So this would mean a probable hazard for vaccine malfunction. (McKinney and Skvoretz 2004)

They believed that the antagonism of the CD4+ Th cell happens due to the decreased number of phosphorylation reactions with the T cell receptor gamma chain. When there are decreased numbers of both CD4+ Th cells and CD8+ T cells, the amount of cytokine produced and the rate at which these cells produce decline. The authors were able to demonstrate that maximal recognition of the variant CD8+ T cell epitopes would often require a "significantly higher concentrations of peptides", that basically suggested "a decreased affinity of TCR binding to these variant forms" (McKinney and Skvoretz 2004) by a process of competitive inhibition.

10) Immunogenicity of clades.

A more recent paper by Santra et al. considers and investigates the difficulties engendered by the genetic diversity of HIV by looking specifically at the amino acid sequences which form the envelopes of viruses. Those from a single subtype can differ by greater than15%, where as those from a different subtype can differ by more than 30% (Santra and Korber et al. 2008). The authors proposed the idea of synthesising immunogens using "centralised HIV-1 gene sequences" which may be used as a solution to this problem. The introduction to the paper offers an overview of the mechanisms used to make centralised genes by a phylogenetic tree: "consensus" - sorting HIV-1 gene chains and then choosing the most prominent amino acid moiety of the virus, "ancestral" - exploring old gene chains or "centre of tree sequences". In essence, this trial compared the breadth of cellular immunity which was generated through immunising rhesus monkeys with vaccines consisting of HIV-1 consensus envelope sequence (CON-S) or either subtype B. The findings of the experiment were that CON-S immunogens could ignite a strong immune activity towards the virus in comparison to subtype B by about 4 times more. The results suggest that this could be a hopeful tactic in developing a vaccine for HIV.

Trialed vaccines

In 2006 AIDSVAX was used in another Phase III trial in combination with ALVAC. It was hoped that a trial combining AIDSVAX, which promotes the production of antibodies to HIV, and ALVAC, which is designed to stimulate a cellular response to the virus, would prove more effective than the previous AIDSVAX trial. The trial recruited 16,402 young adults in Thailand.

The results, published in late 2009, showed that 74 trial candidates who received a placebo became infected with HIV, compared to 51 who had received the vaccine candidate. Although further examination produced mixed results, the analysis which the authors claimed was most relevant showed the vaccine prevented HIV infection by 31.2%. Drawing on this statistically significant result, the authors concluded that the trial showed a "modest protective effect of vaccine".

11) Epidemiology and antigenicity variations of acute and chronic infections

A recent study by Pérez-Losada et al. reports on the result of a Phase-III AIDS Vaccine (VAX004) Trial, a candidate for HIV-1 (AIDSVAX B/B). (Pérez-Losada & Jobes et al. 2010) The main impact of this paper however, is the fact that it documents the largest molecular epidemiologic survey of viruses responsible for new HIV-1 infections in North America.

The authors note that because sequence data from new and recent HIV infections have not previously been available, the selection of vaccine antigens is depends entirely on the sequences of subtype analysis from viruses that come from chronic infections. This study sought to sequence the gp120 envelope sequences of subtype B viruses from 349 recent infections, which represents the most likely basis for the antigenicity of any new infections and therefore is central to considerations of inclusions for the next generation of vaccines. A number of authorities (viz Jobes and Daoust et al. 2006) have reported that the act of infection appears to change the proteins in the HIV coat and therefore the antigenic response to the acute infection may be different from that required to be effective in the chronic infection. This study, although only recently published, represents a huge and valuable resource for the vaccine industry.

12) Significance of acute and chronic infections.

To consider the data from the Pérez-Losada et al. paper further, there is another important finding and that is that the study considered some reasons why the first generation of human immunodeficiency virus vaccines were largely ineffective. They came to the conclusion, largely based on the data from their study that it was due to the fact that viral envelope proteins found in HIV derived from chronic infections appear to have different antigenic structures than envelope proteins from transmission viruses (Pérez-Losada and Jobes et al. 2010). This is borne out by contributory evidence from other studies (viz. Chohan and Lang et al. 2005 & Jobes and Daoust et al. 2006). Therefore in other words, the virus that infects the body, when it enters the cells and replicates, actually produces an antigenically different protein coat when it leaves the cell and moves around the body.

It follows therefore that natural selection plays a role in selecting viruses which cause new infections primarily for infectivity in the absence of an effective immune response, whereas viruses isolated from individuals who have chronic infections have undergone years of selection within the body to evade the immune response. Hypothetically, the authors suggest, the coat may actually be morphologically quite unstable and many different versions are generated from the RNA coding contained within HIV. When the infected cell eventually undergoes apoptosis and releases the newly formed HIV into the blood stream, those that are antigenically the same as the infecting virus will be neutralised by the host immune system and those that are antigenically 'silent' will automatically be selected. This is consistent with the observation by Gilbert et al. that lower viral loads are associated with lower mutation rates. (Gilbert and Rambaut et al. 2007)

Gray et al. have clearly demonstrated in evidence level Ib work, that the immune response to the HIV-1 virus needs to mature for a period in the region of a year or more before antibodies which are capable of broad cross-neutralization are likely to be detected. (Gray and Moore et al. 2007)

Part of the basis of the Pérez-Losada et al. study was that by the assemblage of protein coat sequence data from acute and chronically infected patients will enable investigators to determine whether any fundamental differences that exist in antigenic structure between viruses from recent and chronic infections. It also follows that these sequences will also assist in the assembly of panels of viruses which are representative of new and acute infective agents so that they can be used to evaluate the immunogenicity and potency of future candidate HIV-1 vaccines.

One should note that an epidemiological point also arose from this study and that was that the analysis of HIV subtype B past dynamics suggested that viral populations had already greatly expanded before the number of detected AIDS cases exploded in the 80s when the virus entered the highest risk homosexual population. This gave a baseline that had a huge heterogeneity before serious attempts were made to formulate a vaccine. There were already a wide variety of antigenically distinct human immunodeficiency viruses which were capable of acute infection and this HIV-1 relative genetic diversity has remained almost constant and invariably high ever since this time.

13) Social barriers to vaccine production.

Thus far, this review has considered the technological, biological and immunological barriers to the production of an effective vaccine. A number of authorities (viz. Blankson and Persaud et al. 2002) have pointed to the self-evident fact that, with a world-wide research programme underway, tens of thousands of people, in countries with high levels of HIV, will be required as volunteers for testing. These include the fact that they will have to commit time, be prepared to respond to invasive questions and also potentially face negative social reactions from their family and friends if it is known that they are taking part in such trials. It also follows that the HIV vaccines that they receive may not provide any significant level of protection or equally they may not confer any immediate or long-term benefits. In any pharmaceutical trial, there are obviously inherent clinical risks involved as these are start to be given to people for the first time, even if they may be mainly theoretical rather than practical. (Pilcher and Tien et al. 2008)

The social barriers to this endeavour include the fact that the research in this area is burdened by an inherent public mistrust which is based on being informed wrongly, mistaken theories, and fake expectations largely generated by the media. (USCDCP 2007)

There are also a number of less obvious barriers to progress which, according to French & Gordon include factors such as: a general ignorance of HIV/AIDS, the frequency of messages constantly targeting the community, a lack of basic knowledge of knowing what treatments are available & what is happening in the latest research, denying the existence of HIV/AIDS and the how serious the effects of this disease really are. HIV/AIDS is also given a low priority in communities compared with issues such as jobs and education (French and Gordon et al. 2010)

Discussion and conclusions.

The topic of vaccine production against the human immunodeficiency virus is vast with a huge number of papers being published at an exponential rate. To an extent, this reflects the number of research teams working on the issue, but it also represents an indication of the number of different avenues of exploration that are being considered. This multiplicity of approaches is necessitated by the unique ability of the human immunodeficiency virus to mutate remarkably frequently and thereby temporarily evade the immune system of the human body but it has also developed a number of other mechanisms which allow it to become immunologically silent and to hide the vulnerable trigger sites in its protein coat from the host immune detection and response mechanisms.

To an extent, it is surprising that, in the three decades since the infection reached pandemic proportions, that so much effort has achieved so little success in terms of a functional vaccine. Partly this is because this particular virus is demanding the exploration of new techniques of vaccine operation, but also because it requires the acquisition of new levels of understanding about the pathophysiology engendered by the human immunodeficiency virus.

The groups which initially considered production of a "vaccine" which effectively blocked the uptake of the human immunodeficiency virus into the cell, and thereby prevented infection, were initially optimistic. To a degree their work yielded results but not of a sufficiently consistent nature to offer prophylactic vaccination on this basis. The groups which targeted the chemokine mechanism (CCR5) and those that investigated modified analogues of RATNES had a moderate degree of success to the extent that such techniques are now considered alongside antiretroviral therapy in the treatment of AIDS.

The evidence from these experiences demonstrates that while it is not yet possible to create an immune response which protects every cell in the body from infection, these techniques can slow the progress of cellular infection enough to allow other mechanisms to destroy the infected cells.

Improving the immune response by targeting the CD4+ epitopes appears to have had degrees of success particularly the research aimed at optimising the effect by targeting the evolutionarily highly conserved epitopes which are most likely to be relevant to the highest numbers of human immunodeficiency virus variants. Significant practical problems have been encountered where such immunological enhancement appears to sometimes also adversely affect other T helper cell receptors in an antagonistic manner, thereby reducing the efficacy of the vaccine. Combinations of 10 or more modified epitopes are currently undergoing clinical trials.

Variation in the clade groupings of the amino acid sequences which form the envelopes of viruses are currently one of the most active areas of investigation. He intention being to try to create specific immunogens by using centralised HIV-1 gene sequences to provide a practical solution to the problems of varying immunogenicity.

In essence, there is no vaccine in prospect in the immediate future, although one must acknowledge that huge amounts of research as well as clinical trials are ongoing with a view to expanding the evidence base in this

-In fact, many of the research conducted on HIV envelope proteins did not progress further from phase one, as the antibodies synthesised only neutralised HIV cultured in a lab and not against any wild type HIV strains.

-The authors suggest that using different types of epitopes in just one vaccine, can offer a degree of promise for addressing this issue. Currently clinical trials are underway with potential vaccines which encode for 10 or more epitopes. (Wilson and Newman 2008)