To the client: I am mindful that you want this to be ‘chemistry orientated' and have given this a great deal of thought. The actual chemistry techniques used here are so involved that to describe them in any sort of detail would mean that only a couple of papers would be included in the literature review within the word count and this would not allow examination of the main theme. The title, after all, requires an examination of the problems surrounding the production of an effective vaccine. I have therefore considered a trade off - not only to refer to the chemistry (particularly the biochemistry) behind the research in overview, but to cover as much ground as possible to outline the problems confronting the researchers in this area. PDG

Title: Despite a considerable knowledge of how the AIDS virus works, there is still no effective HIV vaccine, largely due to the Houdini-like properties of the virus itself. What can be done, if anything?

Abstract (summary)

This review considers the background to the development of the HIV/AIDS pandemic and the issues relating to the inherent problems of investigating the production of a vaccine to prevent its spread. The methodology of the review is set out and the review itself is presented in a thematic format.

An overview of the immunogenicity of the human immunodeficiency virus is presented together with the problems that its ability to frequently mutate offer to researchers pursuing the development of a vaccine.

Various insights to the possibility of vaccine production are discussed with a particular prominence given to the chemistry of the various approaches. The feasibility of each technique is critically assessed together with an appreciation of its applicability for the current clinical situation and the implications for future practice.


The first recorded incidence of the human immunodeficiency virus 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 outset but the difficulty in identification was caused by the remarkable ability of the virus to mutate its protein coat, which helps it to evade primary immune system recognition and also its entire genome, primarily through the mechanism of mutations or viral recombination events within gene products. (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 lymphocyte (CTL) and the Th lymphocytes (HTL), it provides a mechanism of escape from immune system control. (Couillin and Connan et al. 1995)

The mainstay of treatment thus far has been the promotion of the "ABC" message of "Abstinence, Be faithful, and use Condoms") by the WHO for prevention and the use of a variety of antiretroviral drugs once the infection has occurred. ( WHO 2005). 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.

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.

Literature Review

Methodology of literature review

Literature reviews as a research methodology

It is fundamentally accepted that the literature review is the starting point for research. Cormack, in his extensive review paper on the mechanics of the research process suggests that “Ultimately all good research is guided by and founded on a critical review of all of the relevant literature published on the subject.” (Cormack, 2000).

It is the critical literature review, which can be presented as either a general or thematic examination of the research area, which is virtually universally regarded as a fundamental, central and vital part of the accumulative process which is the hallmark of the good scientific research project. Other commentators such as Hammond make the observation that not only must original research be ideally placed within a clearly defined and set out historical context so that the reader can identify the process of evolution of the currently accepted beliefs or, for that matter, the derivation of any spectrum of opinion that is presented in the literature, it is also an essential element in the overall research process to try to identify any real or potential gaps in the literature where they can be shown, as Hammond goes on to observe, it is the “identification, exploration and investigation of any of these gaps which are most likely to advance the general understanding and expand the evidence base in that particular area.” Hammond and Howarth 2005 Pg 89)

Hammond also adds that an essential prerequisite to the research process is the process of critical analysis of the material identified as it is the mechanism by which decisions are made which effectively determine whether a study, paper or review needs either further evaluation and research or alternatively it could show that there is a particular weakness in an area of the evidence base of the subject.

Hammond sums up this concept in the verbatim quote “any piece of good quality research should be seen in the context of a continuum which has two major essential components, the literature search, with its attendant processes of critical analysis, as well as the actual writing of the review.” (Hammond, and Howarth 2005 Pg 92)

If an overview of the current literature which is available on the theory of research practices is considered, then it be can determined that there is a wide range of legitimate, useful and often employed research models for conducting an optimally efficient primary literature review.

Most authorities seem to consider that whichever particular model is actually finally chosen for the literature search, it is generally of rather lesser importance than the consideration that the particular process that is finally selected will allow the researcher to arrive at and obtain a consistent and accurate identification and subsequent retrieval of the most relevant and important items from the literature which will allow the researcher not only to glean an overview of the available information which is likely to inform the research process but also to begin to refine the selection of papers that will be ultimately presented in the review.

The identification and retrieval of the relevant studies will also assist in establishing not only the various topics that are likely to be relevant to the particular subject under review but also the associated evidence base for each.

The identification and retrieval of the good quality research that is already available in the literature is of paramount importance, as clearly there is no point in re-exploring areas or duplicating directions of research that have already been the subject of research by others in the field. One obvious and notable exception would be if the research was carried out with the intention of considering whether a challenge to their findings would be worthwhile. (Patton 1990). If one considers the opinion of De Walt, then one might conclude that ”Establishment of a clear picture of what is already known about a particular subject may help to “revisit, change, or even abandon an intended research question so that any subsequent contribution to the evidence base on the subject can be original, useful and informative.” (De Walt and De Walt et al. 1998)

The literature search

It is apparently a common finding for inexperienced researchers to regard the process of a literature search the basic material on a subject to be a linear process which involves a single visit to the data base. Although clearly possible, this type of approach is hardly optimal. In the words of Bowling, it is not a process that “one can rely on to yield optimal search results for the literature review.” (Bowling 2002 pg 22). Bowling offers the novice researcher a number of useful insights in his excellent monograph on the subject with particular regard to searching the literature in an optimal fashion. He suggests that “a good literature search is primarily a cyclical recursive process that mirrors the thinking and research process, where the discovery of new information results in new ideas, new knowledge and possibly new understanding.” (Bowling 2002 pg 24).

Denscombe adds to the debate by suggesting that it is this transfer of the concept of a linear search model to a cyclical and recursive process which is crucial to the integrity and completeness of the final outcome of the form of the literature review as, once the researcher has been able to form an initial overview of the subject area, which includes identification of the major authors and researchers as well as the spectrum of topics that are current, it then enables the researcher to return to initial reviews which were originally accessed and re-read them with a more detailed insight in the subject area, but this process also allows the researcher to then make a better and altogether more fully informed assessment of the data that has been accumulated. (Denscombe 2002)

An additional benefit of this process is that it allows for the initial construction of a thematic grid for the topic under investigation in which each theme that has been identified can then be researched and presented independently of the main topic. This type of thematic review structure is usually considered to be dynamic and can be therefore be modified as the research process evolves and new perspectives become obvious. As new areas become apparent, then they may lead to further, or new themes that can be explored. (Denscombe 2002)

One can also consider the seminal works of Lawrence Carr who has written extensively on the mechanics and processes inherent in constructing a literature review and he focuses on the fact that it is a frequently seen and common fault for researchers who, through ignorance or perhaps lack of initiative, try to restrict their database searching and the subsequent critical analysis of the papers located to one discrete scientific discipline or area. (Carr 1994).

In consideration of this point, if one specifically considerers the major thrust of this particular review topic, then it might be considered that the search for data is most likely to be profitable in journals which are specific to genitourinary medicine, infections diseases or perhaps biochemistry. Even a brief overview of the reference list at the end of this review will show that the information and data has been collected and analysed from across a wide spectrum of scientific disciplines, as well as being extracted from many mutually overlapping subject areas. This method was adopted with the specific intention of trying to assemble and present, a widely informed and well balanced view of the various issues which were pertinent to the topic. A non-exhaustive list of the different research areas which were explored in the literature search for this particular review, although they were not necessarily included and presented in the final presentation of the review article itself, would include areas of research methods, phenomenology, clinical investigation, disease tracking, WHO projects, medicine, virology, clinical trials, biochemistry, immunology, genetics, immunochemistry, sociology, general science, Public Health, microbiology, infectious diseases, epidemiology, chemical pathology, government publications, think tanks, nursing, pathophysiology; demographics; public policy agencies, theoretical research, ethics, legal and legislative, lay media, historical data sources and medical editorials.

Carr's recommendation of expanding the parameters and horizons of the literature search is further supported by Brink's suggestion that, “Each discipline acts, not only as a ‘generator of information' with a specific focus on a particular area of interest, but also as an additional resource to broaden the depth and scope of the resultant paper.” (Brink and Wood 2001). Carr goes on to makes the very astute point that “It is argued that if scholars limit themselves to one method of enquiry, restrictions will be placed on the development of knowledge in their field.” (Carr1994 Pg 726)

Critical appraisal of literature

The essence of a meaningful literature review is not simply the process of locating, reading and re-presenting the facts gleaned from a scientific paper and, according to Schultz, is “a largely sterile and useless process“. (Schulz and Chalmers et al. 2005 Pg 409).

Schultz et al. suggest that what is required is a careful critical analysis and appraisal of both the context and content of each article retrieved and this is the essential and critical element of the whole review process. Schultz also observes that there appears to be a “general and widespread belief that if a particular fact or statement appears in print in reputable peer reviewed literature, then it is sacrosanct and intrinsically unchallengeable. Each article approached and retrieved must be scrupulously and critically appraised for its validity, its reliability and its applicability to the topic in question.” (Schulz and Chalmers et al. 2005 Pg 410)

Thematic analysis of literature findings

One of the first tasks in the preparation of this literature review was the construction of the original list of possible themes for the thematic section of the review. These were initially set out in the very earliest stages of the assembly of the data and they were considered, virtually throughout the entire review process, as a dynamic and actively re-definable entity as the research process progressed.

The initial consideration of the various possibilities of themes generated a considerable number of potential topics that initially appeared suitable for more detailed consideration but, after an initial appraisal, as Moher suggests, as the review process progresses and a more detailed exploration of the extracted material emerges, it becomes entirely appropriate for the researcher to re-define some of the originally designated themes and also to explore a number of alternative topics, areas or directions of further research which may appear to be profitable, or even to re-visit the emphasis that has already been placed on the material that has already been included in the review. This clearly illustrates Bowling's point of a review being a cyclical process, as it may well result in a re-drafting of the initial theme list. (Moher D et al. 1999)

As the literature review is assembled, it is comparatively common to come across conflicting, opposing or overlapping opinions, pronouncements or findings. Far from being a problem, this can be a useful tool for the researcher as considerable insights can be gained and it is clearly of great importance to offer an independent and critical analysis of the evidence base that previous researchers have explored in support of the arguments and expressed views that they have put forward and, in the process, it will allow the author to come to an independent conclusion which is based on consideration of all of the evidence assembled. This process will frequently have the effect of strengthening the evidence base in a particular area and lessons can be learned which may have significance for the current research project.

Bias can be a problem in analysing and presenting data. Any process of critical analysis must include the realisation that it is of prime importance to identify any areas of potential bias which could impact upon the results. Although this tends to be a bigger issue in the quantitative studies, although it is generally less significant in the qualitative ones. (Adèr and Mellenbergh et al. 2008) . If potential areas of bias are found then this has to be translated into a degree of appropriate tolerance in the results when they are finally assimilated into the evidence base. Ahmed has written extensively on the significance of bais and suggests that there may be substantial differences in the study outcome which can arise from even minor anomalies in methodology or quirks in sampling methods. Small variations in the selection criteria for the entry cohort can give rise to variations in the outcome which can affect the statistical significance of the results to the same extent as flaws in the overall design, the methodological process, the execution or the statistical analysis of the study. (Ahmed 2006).

This literature review will be presented in a thematic format which has been constructed in the context of a fluid and dynamic process. The initial selection of potential themes was completed after consideration of the initial literature search. These themes were then explored and any gaps found in the literature were investigated further. Employing a thematic approach also assists the overall process by allowing the bringing together of papers within a specific topic group and thereby making it easier to identify and clarify issues which are not then diluted in the whole review process. In any one particular theme area, the identification of any particularly contentious or disparate opinions is made much easier by the adoption of this methodology. It is the identification of such contentious papers which is of particular importance as these are the ones most likely to act as catalysts for further evaluation or discussion.

An important point is raised by Bell, who observes that it is both appropriate and constructive, when this initial phase is completed, to allocate a short period of time for reflection and consideration of the preliminary results, which will then inform the decisions that are necessary to determine the final shape of the whole review. (Bell 1999)


The methodology which was finally adopted for this literature review was to spend an initial brief period of time to consider and reflect on the various issues which appeared to be obviously central to the main thrust of this review and which appeared to be clearly relevant to the title of the piece. (Denzin and Lincoln, 2000). A small number of representative papers were initially located and read which gave an initial overview of not only the size but also of the quality of the evidence base that was available. This consideration also allowed for an appreciation of both the nature and type of research which had already been undertaken in this area. (Atkins and Murphy 1993). During this initial exploratory phase of the review, a number of lists were drawn up which comprised any references which had been found that might possibly be considered useful in the later phases of the research, together with the names of any authors who appeared to have contributed significantly to the research area in terms of extensive publications in the peer-reviewed literature.

This initial phase concluded with a preliminary drafting of the main research question and the consequent construction of the preliminary thematic structure. The latter was based upon all of the main elements that had been identified in the preliminary work and which seemed to present a useful area for further investigation. This list of initial themes was then correlated with the list of references, authors and papers which had been previously identified. In consideration of the comments by Bowling cited earlier, all of these processes were considered to be both dynamic and fluid and were subsequently modified and updated continuously as the research progressed and as the writing processes evolved.

The actual physical searching of the various databases used was undertaken at local University reference library, the local post-grad clinical library (client to personalise here) and the e-library of the RCP. An amount of additional information, which was not found to be available from these specialist databases was subsequently obtained from a variety of other online databases which were accessed during the process of the review and these included Cochrane, Medline, Hi-wire, Cinhal, Ovid, Questia, The Lancet, The BMJ, the Open University library database and The British Journal Of Nursing,

The search terms that were used included: HIV; AIDS; vaccine; epitope; clade; immune; chemistry; virus; mutation; advances, legal; ethical; These terms were used in various combinations of three terms in a number of searches to sift the database. This process allowed for virtually all of the potentially relevant papers, studies and reviews to be identified within the first seven searches of the database. No significant new papers or studies were identified when subsequent combinations were applied.

An exclusion policy was applied, in general terms, for papers and studies which were published more than 10 yrs ago, unless there was a specific reason by reason of their historical or landmark status to consider their inclusion. (Fink 1998). Occasionally an older paper had to be considered where a particular point needed to be made and a more recent paper of equivalent evidential value could not be identified. (Holliday 2002)

The inclusion criteria that were preferentially adopted were studies and papers which were less than 8 years old and which, after being subjected to a critical appraisal, were judged to have made a substantial and appropriately targeted contribution to the evidence base under consideration. (Punch 2000)

This process resulted in the collection of a large number of papers which were subsequently processed under each of the various thematic headings originally chosen. Each individual paper was then subjected to a process of critical appraisal using the CASP model, (See appendix 2), and was then assigned an appropriate evidence level (See appendix 1). For each point that was to be made, the most significant, recent paper which had the highest evidence level to support that point was then put forward for inclusion into the final draft of the review. (Strauss and Corbin 1990)

During the time that this process was evolving, the major primary themes that were identified in the early stages were subsequently modified. This was primarily due to the application of the processes set out above and which resulted in a re-evaluation of the subject as the review evolved and the final choice of theme was also determined, in part, by the literature that was discovered to be available on the subject. The selection of main themes was finalised and then each theme was fully explored to form the basis of the formal literature review.

Results of the literature search

The search of all of the various databases utilised revealed 162 papers, all of which were subsequently accessed and critically appraised for both the relevance of their content and their evidence level. These 162 papers were then selectively reduced to a total of 29 papers which were considered to have sufficient merit and relevant importance to be suitable for inclusion in the final draft of the literature review. 15 of these papers were then chosen for final inclusion and subsequently presented in varying degrees of depth in the final review .

The mechanism adopted for this subsequent culling was the realisation that some of the papers that were originally selected for inclusion were outclassed by other papers which were judged to have either a greater evidential value or which were more recent in their publication date. Other papers were subsequently discarded because they were considered to be too tangential in their overall content. A number of further papers (a total of 44) were then specifically targeted and accessed in order to offer evidential support for some of the specific points that arose within the body of the review. Other papers were accessed to provide support for some of the points raised by the authors cited in the review. The final decision for the inclusion or the rejection of a particular paper or study was only made after a painstaking consideration of all of the available literature. (Concato and Shah et al. 2000)

The papers and studies which were finally chosen to be included in the literature review comprised a variety of different types of research modality. These included both prospective and retrospective analyses, controlled and non-controlled investigations, several qualitative studies and two authoritative overview review papers together with three meta-analyses.

Thematic Literature review

1) Introduction and historical context

It was clear to many authorities from the outset of the HIV/AIDS pandemic, that it was entirely likely that the human immunodeficiency virus was going to have a global impact. (viz. Mann 1987). This view was clearly vindicated and by 2003 the UN AIDS agency was reporting that in that one year there were approximately five million new HIV infections, while nearly three million people died from AIDS, including approximately 500,000 children under age fifteen. (UNAIDS, 2004).

Taking a complete overview, it is clear that the human immunodeficiency virus 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 also destabilizes the person's family, the community, and the social and economic infrastructure of the global society. In economic terms, the effects of the human immunodeficiency virus are felt in the work force where the pandemic has escalated costs because of absenteeism, sickness, and recruitment; has created organisational disruption and the loss of skills. 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.

HIV/AIDS vaccine. Goals and timeline

The WHO has been championing the development of a human immunodeficiency virus vaccine on the grounds that it would “complement other preventive interventions and could reach populations where other efforts (such as the "ABC" method—"Abstinence, Be faithful, and use Condoms") have been insufficient” ( WHO 2005)

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 most conventional vaccines induce immunity to prevent infection or to keep an infection from persisting and causing disease. The current evidence suggests that the most likely first candidates for a human immunodeficiency virus vaccine will provide only partial protection. (citing Blankson and Persaud et al. 2002).

The current evidence suggests that the first vaccines may initially create an immune response that does not protect every cell in the body from infection but which has the ability to slow the pace of infection enough to allow the natural host defences to rapidly clear virus-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 retrovirals in order to avoid removal. Thus, despite persistent infection, the disease burden and the need for treatment would be diminished. (Catanzaro and Graham 2009)

Elisa techniques

Arguably on 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 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 an antigen is fixed to a non-reactive surface, most usually a polystyrene sheet, and is then washed with a specific antibody and this is allowed to bind with the antigen. After a period for reaction, the substrait plate is gently washed to remove unbonded 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, 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)

Major problems in vaccine development

The HIV-1 virus that causes the vast majority of AIDS cases worldwide, is completely unique in its ability to defeat the body's attempts to create natural immunity. The virus not only mutates very frequently, but it has also developed other mechanisms to become immunologically silent and to hide its vulnerable trigger sites in its protein coat from the host immune responses. It has proved virtually impossible to produce vaccine antigens that can induce broadly neutralising antibodies to HIV. There is no known lasting naturally occurring immunity to HIV/AIDS and 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.

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

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 antibodies from persons actively infected with HIV/AIDS, were able to neutralize infectivity of the human immunodeficiency virus, albeit incompletely with results typically in the region of neutralization of 30 to 50% of human immunodeficiency viruses. (viz. Mascola T R 1996)

By this stage it was known that the human immunodeficiency virus was involved in a complex series of protein-protein interactions in the external cell membrane together which result in structural changes that ultimately result in fusion of the virus and cell membranes and infection of the cell. Specifically, upon binding to CD4, the virus envelope protein undergoes conformational change that allows subsequent interaction with one of several co-receptor molecules, most notably the CC chemokine receptor 5, CCR5, or the CXC chemokine receptor 4, CXCR4. This interaction induces further conformational change in the envelope protein and results in the exposure of the hydrophobic fusion domain of the transmembrane gp41 subunit, which then facilitates fusion of the apposed cell and virus membranes. 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.

This was attempted by culturing with human U87 glioma cells that express CD4 and CCR5 coreceptor (U87-CD4-CCR5) with cells that expressed envelope protein. The process of binding and fusion was studied in progress by stimulating formaldehyde cross-linking before extensive syncytium formation by exposing the culture to 0.2% formaldehyde for 5 hours. This preparation was then used as the fusion-competent (FC) immunogen.

This was inoculated into a mouse model (with transgenic mice that express hu CD4 and hu CCR5 co-receptor) Antibodies generated in this fashion were then demonstrated to have the ability to neutralize the homologous human immunodeficiency virus. This activity was antibody mediated and could therefore be adsorbed to, and subsequently eluted from, a solid support containing protein A and protein G, a mechanism described by Killeen et al. which involves the serum being adsorbed sequentially to protein A Sepharose (Sigma) and protein G agarose (Sigma) at 4°C. The adsorption of antibody being 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. (Killeen and Sawada 1993).

It was hoped that, by targeting the mechanisms that allowed human immunodeficiency virus and sell 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.

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 Hartleya 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. (Hartleya and Gaertnera et al. 2004)

It is known that human immunodeficiency virus entry into mammalian cells requires CD4 plus a chemokine receptor, generally either CXC-chemokine receptor 4 (CXCR4) or CC-chemokine receptor 5 (CCR5), with CCR5 almost exclusively used in transmission and in early stages of disease. (Hartleya and Gaertnera et al. 2004). The importance of CCR5 in the mechanism of HIV transmission appears vital and other evidence strongly suggest that its inactivation would not generate adverse side effects. (Dean and Carrington et al. 2006)

The natural ligands of CCR5 all belong to the chemokine family, which numbers about 40 proteins which are principally involved in control of leukocyte activity, most notably in the modulation of the inflammatory processes and in the maintenance of the adaptive immune response ( Zlotnik and Yoshie 2000) RANTES is one of the most prominent of this family and all are known to inhibit cellular entry of R5-tropic HIV strains, which are defined as those strains requiring CCR5 to enter the cell. (Blanpain and Migeotte et al. 1999)

Hartleya et al point out that it is believed that the chemokines possess structures on the surface of their core domain which are responsible for “address” functions which allow then to dock with receptors with high affinity and specificity, whereas the flexible N-terminal region is responsible for receptor activation. On this basis they sought to synthesise 37 analogues of RATNES , all of which involved incorporation of one or more rationally chosen nonnatural noncoded structures. They screened the analogues for their ability to block the human immunodeficiency virus entry process and succeeded in producing a derivative, PSC-RANTES {N-nonanoyl, des-Ser1[l-thioproline2, l-cyclohexylglycine3]-RANTES(2-68)}, which was 50 times more potent than AOP-RANTES, the first synthetic CCR5 cytokine to be trialed. (Hartleya and Gaertnera et al. 2004)

The chemistry involved in the synthesis of the compounds polymer-supported organic synthesis of two fragments equivalent to the whole proteins after cleavage between residues 33 and 34. t-Butoxycarbonyl (Boc) by the method described by Wilken et al. The various hydrophobic substituents were appended at the N terminus and folding of each synthetic protein with concomitant disulfide formation was carried out in the presence of a Cys-SH/(Cys-S)2 redox couple. (Wilken and Hoover et al. 1999)

Clinical evaluation was done on a severe combined immunodeficient (SCID) (hu-PBL-SCID) mouse model. The mice were injected with the various synthetic compounds including PSC-RANTES, and then challenged with human immunodeficiency virus. The results were highly significant with the treated mice having undetectable (<200 copies per ml) HIV viral RNA for 4 consecutive weeks whereas all infected mice had >10,000 HIV viral RNA copies per ml by week 2 after infection.

This particular paper is presented as being representative of a number of studies which report the application of a high-resolution “medicinal chemistry” approach to the improvement of activity of a synthetic protein. Significant gains in potency were achieved in this study through the rational incorporation of non-natural, (that is non-coded) structures at key sites in existing bioactive proteins to produce a molecule with potential for further clinical development. The authors comment that techniques have now reached the point at which this would be a promising approach to the optimisation of other small proteins of clinical interest.

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 cytotoxic T lymphocyte (CTL) 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+ T help cells are required for the maintenance of CTL and control of viraemia.

In the context of vaccine production, it is thought that the virus might be controlled by a vaccine incorporating improved CD4+ epitopes substituted at some amino acid positions to induce a stronger CD4+ T cell response for helping HIV-specific CTL proliferation, together with similarly enhanced CTL epitopes, 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) CD4 epitope that was the first helper epitope characterised from the human immunodeficiency virus envelope protein (Cease and Margalit et al. 1987) and the same authors showed that immunisation with it is recognized by human T cells from human immunodeficiency virus infected or immunized individuals. Ahlers et al. subsequently showed that potency of this epitope cold be considerably enhanced observed when 436E (glutamic acid) in the T1 epitope is replaced 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+ lymphocytes in humans by exposure to the stimulus of an epitope-enhanced T1 peptide.

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 included the use of subtype-based ancestral or consensus sequences together with the use of highly conserved regions or epitopes (McKinney and Skvoretz 2004)

The rationale behind this approach is that these regions of the HIV-1 genome are believed to be conserved because mutations would negatively impact gene function and general viral fitness. The authors point out that analyses of HIV-1 sequences demonstrate that CTL epitopes are primarily concentrated in the conserved regions, whereas regions that are devoid of CTL epitopes are the most variable (citing Yusim and Kesmir et al. 2002). They also point to the fact that CTL recognition across the various HIV-1 subtypes has been reported in the setting of natural infection and vaccination (citing 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+ T lymphocytes that supports the development of epitope-based vaccines. It is however unfortunately the case that greater than 40% of the sequenced viral isolates contain CTL epitopes from the highly conserved regions of the human immunodeficiency virus genome without amino acid sequence identity to the most prevalent circulating HIV-1 subtypes. (McKinney and Skvoretz 2004)

The methodology of their study was to synthesise epitopes using overlapping oligonucleotides in a PCR-based synthesis in the method set out in the paper by

(Ishioka and Fikes et al. 1999)

The significant feature of this study was the discovery of the fact that some of the epitopes interact with T cell receptor (TCR) in a suboptimal manner can sometimes behave as antagonists and function to actually suppress T lymphocyte responses and this includes the ability of the T lymphocyte to respond to the immunizing epitope. (Klenerman and Rowland-Jones et al. 2004). These antagonistic epitopes are not only the synthetic ones, but some occur naturally. Antagonism of the CD4+ T lymphocyte population has been found in a previous vaccine study (Hartleya and Gaertnera et al. 2004) and is clearly of particular concern because the antagonism was identified in a patient that became infected following vaccination. This mechanism may therefore represent a potential mechanism for vaccine failure. (Oldstone and Tishon 1993)

The McKinney study considered this effect and noted that the antagonism of the T lymphocyte population function is due to reduced levels of phosphorylation of the TCR gamma-chain. Reduced levels of both CD4+ or CD8+ T lymphocyte activation as a result can lead to reduced cytokine production or loss of proliferation and cytolytic capabilities. The authors were able to demonstrate that optimal recognition of the variant CTL epitopes would often require a significantly higher concentrations of peptides, which most likely suggested a decreased affinity of TCR binding to these variant forms by a process of competitive inhibition.

The corollary and clinical importance of these findings is that not only is it the fact that the sequence variation inherent in the HIV-1 gene products which represents one of the most significant obstacles for vaccine development, it is also the fact that some of the epitopes can actively inhibit the T cell response. The authors suggest that it is the use of multiple epitopes in a single vaccine which 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)

Immunogenicity of clades.

A more recent paper by Santra et al. considers and investigates the difficulties engendered by the genetic diversity of the human immunodeficiency virus by looking specifically at the amino acid sequences which form the envelopes of viruses. Those from a single clade can differ by >15%, and those from different clades can differ by >30%. (Gaschen B et al.2002). The authors proposed that creating immunogens using centralised HIV-1 gene sequences might be harnessed to provide a practical solution to this difficulty. The introduction to the paper offers an overview of the mechanisms used to generate the centralised genes such as consensus, ancestral, or centre of tree sequences. In essence, this trial compared the breadth of cellular immunity which was generated through immunisation of rhesus monkeys with vaccine constructs which expressed either an HIV-1 global consensus envelope sequence (CON-S) with that expressed by a single patient isolate clade B envelope sequence (clade B). The outcome was that vaccine immunogens which expressed a single centralized gene, CON-S, was able to generate cellular immune responses which had significantly increased breadth of activity when compared with immunogens which expressing a wild-type virus gene. This difference was found to be that the CON-S immunogens elicited cellular immune responses to 3- to 4-fold more epitopes of the viral envelope proteins from clades A, C, and G than did clade B immunogens. The significance of these findings are that it seems likely that immunisation with centralised genes could be promising vaccine strategy for developing a global vaccine for the human immunodeficiency virus.

Epidemiology and antigenicity variations of acute and chronic infections

As a final contribution, one can consider the very recent Pérez-Losada et al. study which reports on the result of a Phase-III AIDS Vaccine (VAX004)

Trial. (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 human immunodeficiency virus infections have not previously been available, the selection of vaccine antigens is dependent upon sequences of clade analysis from viruses from chronic infections. This study sought to sequence the gp120 envelope sequences of clade B viruses from 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 human immunodeficiency virus 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.

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 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 area.

Appendix 2


Quantitative Research Papers Critiquing Tool

A Framework for Critiquing Quantitative Research Papers Include full reference of paper here: (i.e. Author/s (date) Title of article. Journal title. Volume, Number, page numbers.) Critiquing Framework Title of Paper Is the aim or purpose of the study clear? Are the main variables of interest indicated? Is the study design or research method clear from the title? Is there any reference to the population from whom the data are collected?

The Abstract/Summary Does this summarise the whole study? Is information provided regarding background, literature, aim/and objectives, hypotheses (if RCT), methods, sample size, measures used etc, results and conclusions? Does it suggest that a more detailed reading of the rest of the paper would be worthwhile?

Introduction/Literature Review Background/rationale Why was the topic chosen, what is the background to the study? Is there a critical review of previous literature and related theoretical concepts? Are gaps in the literature identified? Aim and objectives What problem or issue is being investigated? How clearly is this problem or issue defined or explained? Is there a clearly stated aim? Do the research objectives or research questions support this aim? Are the variables of interest clearly defined `and are relationships between these evident and clearly stated? Which are the independent and dependent variables? Are hypotheses (if RCT) stated in a way that makes them testable?

Method Research design What is the study design and is it clearly explained and appropriate for the research questions? Could the design be improved? Was there a pilot study?

Research questionnaires What measures are used? Are validity and reliability reported for these measures either in the paper or clearly referenced? Have the authors dealt appropriately with any unreliable questionnaires or scales?

Sample Is the population appropriate for the research question? How were the sample chosen? What is the sample size? Are statistical power considerations discussed? Are inclusion and exclusion criteria described? Can the results be reasonably generalised on the basis of this sample?

Ethics Are ethical considerations presented. Is it suggested that ethical approval was granted?

Results/Findings/Data analysis Does the paper explain clearly how the data are analysed? Are statistical techniques clearly and adequately described? Are the statistics presented at a simple descriptive level or are inferential statistics also included? How are the results presented? Does the text adequately explain any tables or graphs? Have any tests of significance established whether differences, or associations, between groups could have happened by chance? What p values are used? Are non-significant results clearly indicated?

Discussion Is the discussion an accurate account of the results? Could there be other ways of interpreting the data? Does the discussion address the research aim and objectives? Are all the research questions answered?

Conclusion Are the conclusions of the study consistent with the results of the statistical analyses? Are alternative conclusions suggested? Are theoretical and practical implications of the results adequately discussed? Are the recommendations suggested feasible?

Limitations What are the limitations and are these acknowledged by the authors? Overall impression

(CASP 2000)


Adèr, H. J., Mellenbergh, G. J., & Hand, D. J. (2008). Advising on research methods: A consultant's companion. Huizen, The Netherlands: Johannes van Kessel Publishing.

Ahlers, J. D., I. M. Belyakov, E. K. Thomas, J. A. Berzofsky. (2001). High affinity T-helper epitope induces complementary helper and APC polarization, increased CTL and protection against viral infection. J. Clin. Invest. 108 : 1677 - 1685.

Ahmed, S. (2006) Queer Phenomenology : Orientations, Objects Others. Durham: Duke University Press. 2006

Asjö, B., F. Barin, G. Biberfeld, J. Bradac, A. Buvé, S. Dielly, A. Fontanet, L. Gurtler, G. Van der Groen, M. Hoelscher, et al (1997). HIV - 1 subtypes: implications for epidemiology, pathogenicity, vaccines and diagnostics: workshop report from the European Commission (DG XII, INCO - DC) and the Joint United Nations Programme on HIV/AIDS. AIDS 11 : A17.

Atkins, S. & Murphy, K. (1993) Reflection : A Review of the Literature. Journal of Advanced Nursing. 18 (8), 1188 - 1192.

Bell J (1999) Doing your research project. A guide for first time researchers in education and social science. 3rd edition. Buckingham; Open University Press

Berzofsky, J. A., J. D. Ahlers, I. M. Belyakov. (2001). Strategies for designing and optimizing new generation vaccines. Nat. Rev. Immunol. 1 : 209 - 219.

Blankson J N, Persaud D, Siliciano R F (2002) , "The Challenge of Viral Reservoirs in HIV-1 Infection," Annual Review of Medicine 53 (2002) : 557 - 593

Blanpain, C., Migeotte, I., Lee, B., Vakili, J., Doranz, B. J., Govaerts, C., Vassart, G., Doms, R. W. & Parmentier, M. (1999) CCR5 Binds Multiple CC-Chemokines: MCP-3 Acts as a Natural Antagonist. Blood 94 , 1899 - 1905

Brink, P. J. & Wood M. J. (2001) Basic steps in planning nursing research: from question to proposal. 5th ed. London; Jones and Bartlett. 610.73072

Carr LT (1994) The strengths and weaknesses of quantitative and qualitative research: what method for nursing? Journal of Advanced Nursing Volume 20 Page 716 - October 1994

CASP (2000), Critical Appraisal Skills Programme cited in Taylor R, Barnaby Reeves, Paul Ewings, Sarah Binns, John Keast, Rebecca Mears (2000) A systematic review of the effectiveness of critical appraisal skills training for clinicians. Medical Education 34 (2) , 120 - 125

Catanzaro A T Graham B S (2009) , "Rationale for Current HIV Clinical Trials," in Recent Advances in HIV Infection Research, ed. G. Buela-Casal and M.P. Bermudez Granada, Spain: University of Granada,

Cease, K. B., H. Margalit, J. L. Cornette, S. D. Putney, W. G. Robey, C. Ouyang, H. Z. Streicher, P. J. Fischinger, R. C. Gallo, C. DeLisi, J. A. Berzofsky. (1987). Helper T cell antigenic site identification in the AIDS virus gp120 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide. Proc. Natl. Acad. Sci. USA 84 : 4249 - 4253

Christiansen C B, Jessen T E, Nielsen C, Staun-Olsen P. (2006) False negative anti-HIV-1/HIV-2 ELISAs in acute HIV-2 infection. Vox Sang 2006 ; 70 : 144 - 147

Concato, J, Shah, N, Horwitz, RI (2000) Randomised, controlled trials, observational studies, and the hierarchy of research designs. N Engl J Med 2000 ; 342 , 1887 - 1892

Cormack, D. (2000) The Research Process in Nursing : Fourth Edition. London: Blackwell Science. 2000

Couillin, I., F. Connan, B. Culmann-Penciolelli, E. Gomard, J. G. Guillet, J. Choppin. 1995. HLA-dependent variations in human immunodeficiency virus Nef protein alter peptide/HLA binding. Eur. J. Immunol. 25 : 728 - 9

Dean, M., Carrington, M., Winkler, C., Huttley, G. A., Smith, M. W., Allikmets, R., Goedert, J. J., Buchbinder, S. P., Vittinghoff, E., Gomperts, E., et al. (2006) Genetic Restriction of HIV-1 Infection and Progression to AIDS by a Deletion Allele of the CKR5 Structural Gene Science 273 , 1856 - 1862.

Denscombe M N (2002) Ground rules for good research. Maidenhead, Open University Press.

Denzin, N K. & Lincoln, Y S. (2000) Handbook of Qualitative Research : (second edition), London; Sage.

DeWalt, K. M., DeWalt, B. R., & Wayland, C. B. (1998). Participant observation. In H. R. Bernard (Ed.) Handbook of methods in Cultural Anthropology. (pp. 259 - 299). Walnut Creek, CA : Alta Mira Press. 1998

Eccles, M. and Mason, J. (2001) How to develop cost-conscious guidelines. Health Technology Assessment 5 (16), 1 - 78.

Esparza J, Bhamarapravati N (2000) "Accelerating the Development and Future Availability of HIV-1 Vaccines: Why, When, Where, and How?" Lancet 355, no. 9220 (2000) : 2061 - 2066

Fink A (1998) Conducting research literature reviews: from paper to the internet Sage, London

Fukada, K., H. Tomiyama, C. Wasi, T. Matsuda, S. Kusagawa, H. Sato, S. Oka, Y. Takebe, M. Takiguchi. (2002). Cytotoxic T-cell recognition of HIV-1 cross-clade and clade-specific epitopes in HIV-1-infected Thai and Japanese patients. AIDS 16 : 701 - 3

Gaschen B et al. (2002) Diversity considerations in HIV-1 vaccine selection. Science 296 : 2354 - 2360

Glynn J R, Biraro S, Weiss H A. (2009) Herpes simplex virus type 2: a key role in HIV incidence. AIDS 2009; 23 : 1595 - 1598.

Hammond, M., Howarth, J. & Kent, R. (2005) Understanding Phenomenology.

Oxford : Blackwell. 2005

Hartleya O, Gaertnera H, Wilkend J, Thompsond D, Fisha R, Ramosh R et al. (2004). Medicinal chemistry applied to a synthetic protein: Development of highly potent HIV entry inhibitors. PNAS November 23, 2004 vol. 101 no. 47 16460 - 16465

Hasenkrug, K. J., D. M. Brooks, U. Dittmer. (1998). Critical role for CD4+ T cells in controlling retrovirus replication and spread in persistently infected mice. J. Virol. 72 : 6559 - 6564.

Heyward W L, MacQueen K M, Goldenthal K L. (1998) "HIV Vaccine Development and Evaluation: Realistic Expectations," AIDS Research and Human Retroviruses 14, Supp. 3 (1998) : S205 - S210.

Holliday A (2002) Doing and writing qualitative research. London; Sage Publications

Ishioka, G. Y., J. Fikes, G. Hermanson, B. Livingston, C. Crimi, M. Qin, M. F. del Guercio, C. Oseroff, C. Dahlberg, J. Alexander, et al (1999) . Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes. J. Immunol. 162 : 3915 - 20

Jobes D V, Daoust M N, Nguyen V D, Padua A P, Michele S O, Lock M D, Chen A P, Sinangil F, Berman P W. (2006) High incidence of unusual cysteine variants in gp120 envelope proteins from early HIV type 1 infections from a phase 3 vaccine efficacy trial. AIDS Res Hum Retrovir (2006) 22 : 1014 - 1021

Killeen N, Sawada S, Littman D R, (1993) EMBO J. 12, 1547 (1993)

Klenerman, P., S. Rowland-Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, et al (1994). Cytotoxic T-cell activity antagonized by naturally occurring HIV-1 Gag variants. Nature 369 : 403

LaCasse R A, Follis K E, Trahey M N, Scarborough J D, Littman D R, Nunberg J H (1999) Fusion-Competent Vaccines: Broad Neutralization of Primary Isolates of HIV/AIDS. Science 15 January 1999 : Vol. 283. no. 5400, pp. 357 - 362

Mann J M (1987), "The Global AIDS Situation," World Health Statistics Quarterly 40, no. 2 (1987) : 185 - 192.

Mascola T R (1996), et al., AIDS Res. Hum. Retrovir. 12, 13198 (1996) ; J. P. Moore, et al., J. Virol. 70, 427 (1996)

McKinney D M, Skvoretz R, Livingston B D, Wilson C V, Anders M, Chesnut R W et al. (2004) Recognition of Variant HIV-1 Epitopes from Diverse Viral Subtypes by Vaccine-Induced CTL. The Journal of Immunology, 2004, 173 : 1941 - 1950.

Moher D, Cook D J, Eastwood S, Olkin I, Rennie D, Stroup D F, for the QUOROM Group. (1999) Improving the quality of reports of meta-analyses of randomised controlled trials : the QUOROM statement. Lancet 1999; 354 : 1896 - 1900

Okazaki T, Pendleton C D, Sarobe P, Thomas E K, Iyengar S, Harro C, Schwartz D, Berzofsky J D (2006) Epitope Enhancement of a CD4 HIV Epitope toward the Development of the Next Generation HIV Vaccine. The Journal of Immunology, 2006, 176 : 3753 - 3759.

Oldstone, M. B. A., A. Tishon, M. Eddleston, J. C. De la Torre, T. McKee, J. L. Whitton. (1993) . Vaccination to prevent persistent viral infection. J. Virol. 67 : 4372

Pandori M W, Hackett J K, Louie B N, Vallari A S, Dowling T D, Liska S T, Klausner J D (2009) Assessment of the Ability of a Fourth-Generation Immunoassay for Human Immunodeficiency Virus (HIV) Antibody and p24 Antigen To Detect both Acute and Recent HIV Infections in a High-Risk Setting. J. Clin. Microbiol., August 1, 2009; 47(8): 2639 - 2642.

Patton, M Q. (1990) Qualitative Evaluation and Research Methods. Thousand Oaks, California, USA : Sage Publication

Pérez-Losada M N, Jobes D V, Sinangil F P, Crandall K A, Posada K P, Berman P W (2010) Phylodynamics of HIV-1 from a Phase-III AIDS Vaccine Trial in North America. Molecular Biology and Evolution 2010 27 (2) : 417 - 425;

Pilcher C D et al. (2004) "Brief but Efficient: Acute HIV Infection and the Sexual Transmission of HIV," Journal of Infectious Diseases 189, no. 10 (2004) : 1785 - 1792.

Punch, K. F. (2000) Developing effective research proposals. London; Sage. 300 . 72 - 75

Santra S A, Korber B T, Mul;doon M N, Barouch D H, Nabel G J, Gao F G, Hahn B H, Haynes B F, Letvin N L (2008) A centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys. PNAS July 29, 2008 vol. 105 no. 30 10489 - 10494

Schulz K F, Chalmers I, Hayes R J, Altman D G. (2005) Empirical evidence of bias. Dimensions of methodological quality associated with estimates of treatment effects in controlled trials. JAMA 2005; 273 : 408 - 412

Strauss A & Corbin J (1990) Qualitative Research Methods. Thousand Oaks, Sage.

UNAIDS, (2004) Report on the Global AIDS Epidemic (Geneva: Joint United Nations Programme on HIV/AIDS, June 2004), 10.

van der Graaf M, Diepersloot R J. (1986) Transmission of human immunodeficiency virus (HIV/HTLV-III/LAV): a review. Infection 14, 203 - 11

WHO (2005) World Health Organization, "HIV Vaccines," (accessed 19.3.2010).

Wilken, J., Hoover, D., Thompson, D. A., Barlow, P. N., McSparron, H., Picard, L., Wlodawer, A., Lubkowski, J. & Kent, S. B. (1999) Total chemical synthesis and high-resolution crystal structure of the potent anti-HIV protein AOP - RANTES. Chem. Biol. 6 , 43 - 51. Pmid : 9889 - 151

Wilson C D, Newman M N, Livingston B D, MaWhinney S D, Forster J E, Scott J P, Schooley R T, Benson C A (2008) Clinical Phase 1 Testing of the Safety and Immunogenicity of an Epitope-Based DNA Vaccine in Human Immunodeficiency Virus Type 1-Infected Subjects Receiving Highly Active Antiretroviral Therapy. Clin. Vaccine Immunol., June 1, 2008; 15 (6) : 986 - 994.

Yusim, K., C. Kesmir, B. Gaschen, M. M. Addo, M. Altfeld, S. Brunak, A. Chigaev, V. Detours, B. T. Korber. (2002). Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation. J. Virol. 76 : 8757 - 60.

Zhu T, Korber B T, Nahmias A J, Hooper E, Sharp P M, Ho D D. (1998) An African HIV-1 sequence from 1959 and implications for the origin of the epidemic. Nature 391, 594 - 597

Zlotnik, A. & Yoshie, O. (2000) Chemokines: a new classification system and their role in immunity. Immunity 12 , 121 - 127. Pmid : 1071 - 4678

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