Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 2, 2002, pp. 143-150

 


Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(2) 2002, pp. 143-150
HIV-1 Polymorphism: a Challenge 
  for Vaccine Development - A Review 
MG Morgado/+, ML Guimarães, 
  B Galvão-Castro*  
Laboratório de AIDS e Imunologia 
  Molecular, Departamento de Imunologia, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 
  4365, 21045-900 Rio de Janeiro, RJ, Brasil *Laboratório Avançado de Saúde Pública, 
  Centro de Pesquisa Gonçalo Moniz-Fiocruz, Salvador, BA, Brasil 

  +Corresponding author. Fax: +55-21-2280.1589. E-mail: mmorgado@ioc.fiocruz.br  
Fiocruz is an UNAIDS Collaborative 
  Centre. 

  This work was supported by the National Coordination of STD/AIDS/UNDP/World 
  Bank, (UNAIDS), PIAF/Fiocruz/Ministry of Health, Faperj and Brazilian Research 
  Council (CNPq). 

  

  Received 5 January 2002 

  Accepted 15 February 2002 
Code Number: oc02028 
The perspective for the development 
  of anti-HIV/AIDS vaccines became a target sought by several research groups 
  and pharmaceutical companies. However, the complex virus biology in addition 
  to a striking genetic variability and the limited understanding of the immunological 
  correlates of protection have made this an enormous scientific challenge not 
  overcome so far. In this review we presented an updating of HIV-1 subtypes and 
  recombinant viruses circulating in South American countries, focusing mainly 
  on Brazil, as one of the challenges for HIV vaccine development. Moreover, we 
  discussed the importance of stimulating developing countries to participate 
  in the process of vaccine evaluation, not only testing vaccines according to 
  already defined protocols, but also working together with them, in order to 
  take into consideration their local information on virus diversity and host 
  genetic background relevant for the vaccine development and testing, as well 
  as including local virus based reagents to evaluate the immunogenicity of the 
  candidate vaccines.  
 
Key words: HIV-1 polymorphism - HIV/AIDS 
  - vaccine  
Since the identification of the viral 
  etiology of the Acquired Immunodeficiency Syndrome (AIDS) in 1983, with the 
  isolation of the Immunodeficiency Virus Type 1 (HIV-1) from patients with persistent 
  lymphadenopathy (Barre-Sinousi et al. 1983), the perspective for the development 
  of anti-HIV/AIDS vaccines became a target sought by several research groups 
  and pharmaceutical companies. However, the complex virus biology in addition 
  to a striking genetic variability and the limited understanding of the immunological 
  correlates of protection have made this an enormous scientific challenge not 
  overcome so far.  
Indeed, HIV is one of the most important 
  emergent pathogens of this century. Based on phylogenetic analyses, it was estimated 
  to have emerged in the human population around 1931 (Korber et al. 2000), becoming 
  a worldwide public health challenge fifty years later.  
As recently reviewed by the Joint 
  United Nations Programme on HIV/AIDS (UNAIDS 2001), since the description of 
  the first cases at the early beginning of the eighties in USA and Europe, more 
  than 21,8 million deaths have occurred due to HIV/AIDS. Forty million individuals, 
  adults and children, were estimated to be living with HIV/AIDS worldwide by 
  the end of 2001 and Latin America contributes with almost 4% of these cases. 
  HIV/AIDS was the cause of 4.2% of deaths in the world in 1999 being the fifth 
  cause of death worldwide. Only in 2001, 5.3 million adults and children were 
  estimated to be infected worldwide, with 14,000 new infections a day. Around 
  95% of these new cases occurred in developing countries. Roughly 14% were in 
  children under 15 and 86% in people aged 15-49, of whom almost 50% are women 
  and about 50% are 15-24 years old.  
Although the development of combined 
  antiretroviral therapy has had a great impact on the reduction of AIDS morbidity 
  and mortality, its application is quite limited worldwide due to the high cost 
  and complex medical approach for the more deprived areas, complicated by the 
  emergence of drug resistant strains. Thus, the development of HIV/AIDS vaccine 
  is of high priority for public health.  
There is a newly growing optimism 
  in the international scientific community concerning the possibility of developing 
  safe and effective preventive vaccines. However, despite the progress in recent 
  years, one of the major points is still to be clarified concerning the importance 
  of the intriguing virus diversity on vaccine efficacy. Indeed, viruses isolated 
  from different patients, and especially from different geographic regions, show 
  considerable genetic and thus antigenic variations, which may be a limiting 
  factor to the development of a universal vaccine.  
MOLECULAR EPIDEMIOLOGY 
  OF HIV  
The molecular epidemiology of HIV 
  and its potential implications for vaccine development and efficacy, as well 
  as for other aspects of HIV infection such as local epidemiological features, 
  virus transmission and pathogenesis, has been an issue of great concern (reviewed 
  by Workshop Report 1997a,b). Indeed, HIV is a highly polymorphic virus and two 
  types, HIV-1 and HIV-2, are participating in the AIDS epidemic. While HIV-2 
  has been basically restricted to the African continent, HIV-1 is associated 
  with the worldwide pandemic. Phylogenetic analyses of virus samples from different 
  geographic regions have revealed that HIV-1 can be divided into three groups: 
  O (outlier), N (new, non-N, non-O) and the M group (Major). While samples from 
  groups O and N are limited to the African continent, with some isolated cases 
  of group O in Europe and in the United States, group M variants are responsible 
  for the AIDS pandemic and were found to be subdivided into major genetic forms, 
  including distinct subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, 
  J and K). Moreover, 14 circulating recombinant forms (CRF 1 to 14), in addition 
  to unique recombinant viruses and several other HIV-1 unclassified genomes have 
  already been identified (reviewed in McCutchan 2000, CRF homepage http://hiv-web.lanl.gov/ for continuous 
  updating), as well as mixed infections (Janini et al. 1996, 1998, Ramos et al. 
  1999, Flores et al. 1999). This striking worldwide genetic variability is depicted 
  in the Figure.  
The HIV-1 subtypes are geographically 
  distributed, with multiple subtypes and recombinant viruses co-circulating in 
  many areas of the world. All of them are similarly transmitted through sexual 
  contact, blood contamination, and vertically from mother to child.  
Pairwise nucleotide distance among 
  the HIV-1 subtypes showed 20% for the envelope, 16% for the gag and 13% for 
  the polymerase genomic regions (Robertson et al. 1999). Such a high genetic 
  variability has direct influence on the antigenic constitution of these viruses. 
  Indeed, comparative analyses of relevant B cell, CD4+ T helper cell 
  and cytotoxic CD8+ T-lymphocytes (CTL) epitopes located in these 
  regions have shown an important level of variability in a greater or lesser 
  extent among the HIV-1 subtypes (HIV Molecular Immunology 2000).  
It is therefore important to know 
  whether the HIV-1 strains circulating in the countries where the vaccine will 
  tentatively be used, are genetically and antigenically related to the vaccine 
  prototype being tested. In order to obtain this type of information, it is necessary 
  to keep HIV molecular surveillance programs, checking systematically the distribution 
  of the HIV-1 subtypes, as well as the introduction of new variants.  
With the perspective of HIV/AIDS 
  preventive vaccine trials at the beginning of the nineties to be conducted in 
  different world regions, in both developed and developing countries, the knowledge 
  of the molecular epidemiology of HIV rapidly increased worldwide (Figure) and Brazil was one of the countries 
  participating in this international effort.  
Several studies on the molecular 
  epidemiology of HIV-1 in Brazil showed the predominance of subtype B, with the 
  presence of additional subtypes like F (Morgado et al. 1994, 1996, 1998a, Galvão-Castro 
  et al. 1996, Tanuri et al. 1999, Brazilian Network for HIV Isolation and Characterization 
  2000), C (WHO 1994, Loureiro et al. 1998, Antunes 1998), D (Morgado et al. 1998a, 
  Tanuri et al. 1999) and, more recently, the presence of subtype A (Caride et 
  al. 2001, Guimarães et al. unpublished). Recombinant B/F and B/C viruses have 
  also been described (Sabino et al. 1994, Gao et al. 1996, Cornelissen et al. 
  1996, Tanuri et al. 1999, Vicente et al. 2000, Guimarães et al. 2001), in addition 
  to dual subtype infections (Janini et al 1996, 1998, Ramos et al. 1999).  
Due to the interest for vaccine purposes, 
  the majority of these studies were originally performed based on the analysis 
  of the envelope gene, and, more recently, the evaluation of gag and polymerase 
  genomic polymorphisms have already been assessed. In addition to the inter-subtype 
  diversity, genetic and antigenic differences have also been described among 
  the subtype B viruses circulating in Brazil, with the identification of a subtype 
  B variant called B", which differs from the classical subtype B viruses by the 
  presence of the GWGR motif at the top of the envelope gp120 V3 loop, instead 
  of the GPGR molecular signature (Potts et al. 1993, Morgado et al. 1994, Brazilian 
  Network for HIV Isolation and Characterization 2000). These two subtype B variants 
  can be distinguished by both genetic (Morgado et al. 1998b, Covas et al. 1998) 
  and antigenic (Bongertz et al. 1994, Hendry et al. 1996, Casseb et al. 1998) 
  approaches, although consistent cross-neutralization has been observed between 
  them (Bongertz et al. 1997, 1998). Such observations are of important consideration 
  for vaccine evaluation in our country as the B" subtype B variant was found 
  to be highly prevalent in some Brazilian areas, corresponding to 57% of the 
  subtype B samples detected in Ribeirão Preto, SP (Covas et al. 1998) and 37% 
  of those from Rio de Janeiro, RJ (Morgado et al. 1998a,b).  
Brazil is a huge country and differences 
  in the pattern of subtype distribution have been identified among the geographic 
  regions. Indeed, whereas similar proportions of B and F subtypes were observed 
  in a study conducted in Manaus (northern Brazil) (Vicente et al. 2000), in the 
  southeast, several studies have shown the predominance of subtype B (~85%) followed 
  by subtype F (~10-15%) (Morgado et al. 1994, 1998a, Tanuri et al. 1999, Brazilian 
  Network for HIV Isolation and Characterization 2000). Moreover, a clear predominance 
  of subtype B (> 90%), with very limited cases of F and C subtypes were, respectively, 
  observed in HIV-1 samples from the northeast (Couto-Fernandez et al. 1999) and 
  central-west (Stefani et al. 2000). The presence of subtype C in south Brazil 
  was firstly detected in 1 out 5 HIV-1 samples collected in Porto Alegre, RS, 
  in a setting of a WHO international study (WHO 1994). In a recent analysis conducted 
  by our group (unpublished data), we observed in 34% of the HIV-1 subtype C infected 
  samples collected in Porto Alegre, RS, in 1998, strongly contrasting with the 
  pattern of HIV-1 subtype distribution already verified for other Brazilian geographic 
  regions. This subtype seems to be spreading all over the world and is presently 
  the most prevalent strain in the AIDS pandemic (Esparza & Bhamarapravati 
  2000).  
Following the Brazilian pattern, 
  studies on the molecular epidemiology of HIV-1 in other South American countries 
  also showed the predominance of subtype B, with the presence of additional subtypes 
  like F in Argentina, Bolivia, Uruguay, Peru, Paraguay, and Venezuela (Campodonico 
  et al. 1996, Marquina et al.1996, Velarde-Dunois et al. 2000, Masciotra et al. 
  2000, Russel et al. 2000, Castro et al. unpublished) and isolated cases of A 
  in Chile (Desgranges et al. 1998), Peru (Russel et al. 2000) and French Guyana 
  (Kazanji et al. 2001) and E in Uruguay (Artenstein et al. 1995). Moreover, unique 
  recombinant B/F viruses and/or CRF_012 B/F have also been described in Argentina 
  (Marquina et al. 1996, Fernandez-Medina et al.1999, Carr et al. 2001), Uruguay 
  (Carr et al. 2001) and Venezuela (Delgado et al. 2001, Castro et al. 2001). 
  Data from Colombia and Ecuador have only shown the presence of subtype B so 
  far (Navas et al. 1999, Russel et al. 2000).  
The general picture of the HIV-1 
  subtypes and recombinant viruses circulating in South American countries is 
  summarized in the Figure. In general, a quite similar virus 
  pattern distribution is observed among the countries, which certainly will be 
  reflected in similar HIV/AIDS vaccine strategies to be applied for South America. 
   
HIV VACCINE CANDIDATES 
The first generation of HIV vaccine 
  concepts favored the protective humoral immune response, characterized by the 
  presence of neutralizing antibodies mainly to the V3 loop of the viral envelope. 
  The first Phase I protocol, corresponding to a gp160 recombinant protein started 
  in 1987, less than 5 years after the virus identification (Dolin et al. 1991). 
  Several other protocols were followed also focusing on the envelope gp120 or 
  the full-length gp160, produced in different cell systems by distinct vaccine 
  developer initiatives (reviewed by Bojak et al. 2002). Indeed, the first HIV-1 
  vaccine presently in a Phase III efficacy trial corresponds to this vaccine 
  concept (Francis et al. 1998). This efficacy trial is now being conducted in 
  the USA and Thailand using, respectively, bivalent HIV-1 gp120 B/B and B/E subtype 
  vaccines (Berman et al. 1999), and the first results are expected soon. However, 
  the difficulty to neutralize primary isolates of the virus was always a limiting 
  factor of these vaccine concepts (Mascola et al. 1996), which are not able to 
  induce antibodies against broadly conserved neutralizing epitopes (reviewed 
  by Moore et al. 2001). Although there is no correlation between HIV-1 subtypes 
  and virus neutralization, cross-clade neutralization has been described in a 
  greater or lesser extent by several authors (reviewed by Moore et al. 2001), 
  thus reflecting the presence of common antibody epitopes, at least among groupings 
  of HIV-1 subtypes and variant viruses.  
A highly relevant factor described 
  in recent years by many research groups was the association between the presence 
  of HIV-1 specific cytotoxic CD8+ T-lymphocytes (CTL) and the decrease 
  of the plasma viral load in HIV infection (reviewed by Brander & Walker 
  1999). Indeed, a negative correlation was demonstrated between the frequency 
  of CTL and progression to disease in HIV infected individuals (Klein et al. 
  1995, Musey et al. 1997). Also, dramatic increases in viral load were observed 
  in chronically SIV-infected macaques after CD8+ T cell depletion treatment, 
  which coincidentally decreases with the reappearance of CD8+ cells (Jin et al. 
  1999, Schmitz et al. 1999). These findings have certainly strengthened the importance 
  of the cell-mediated immunity in the control of the HIV infection and disease 
  progression, leading to a redirection of the HIV vaccine protocols for the utilization 
  of live vectors able to induce MHC class I presentation of processed antigenic 
  peptides to CD8+ T-lymphocytes. Furthermore, broad cross-reactive CTL recognition 
  has been demonstrated in relation to the CTL epitopes located in very conserved 
  regions of HIV-1 genome among the HIV-1 subtypes (Ferrari et al. 1997, Cao et 
  al. 1997, McAdam et al. 1998), although reactions of higher magnitude have been 
  described for subtype-specific CTL responses (Rowland-Jones et al. 1998, Cao 
  et al. 2000, Novitsky et al. 2001).  
In this sense, recombinant pox viruses 
  and DNA vaccine concepts have already been tested in Phase I clinical trials. 
  Promising alternative vector-based approaches, like recombinant poliovirus, 
  Venezuelan equine encephalitis virus (VEE), BCG, salmonella and shigella, among 
  others, are under development for HIV vaccines (reviewed by Bojak et al. 2002). 
   
If on one hand, no specific CTL responses 
  were observed in the volunteers vaccinated with HIV-1 recombinant protein vaccine 
  protocols, on the other hand, limited neutralizing antibody production was also 
  observed when recombinant pox virus vaccines were administered alone in human 
  HIV-1 vaccine trials (Mulligan & Weber 1999). Considering the relevance 
  of both humoral immune response and cell mediated immunity on HIV control, more 
  promising vaccine approaches were further proposed, denominated as prime-boost 
  regimen including vaccine concepts with complementary immunogenic profiles, 
  associated with the inclusion of multi-antigen components consisting of selected 
  HIV-1 structural as well as regulatory antigen-encoding genes. Together, these 
  types of vaccine approaches are attempting to amplify the immune response to 
  the virus, inducing a multi-specific, long lasting humoral and cell mediated 
  immunity able to prevent HIV infection and/or progression to disease.  
Since the first human trial of HIV-vaccine 
  at the end of the eighties, around 30 different HIV-1 candidate vaccines were 
  tested in more than 60 phase I/II clinical trials (see Bojak et al. 2002 for 
  updating). The majority of them have been conducted in USA and Europe, but a 
  few others have already been conducted in developing countries like Thailand, 
  China, Cuba, Uganda and Brazil (Esparza & Bhamarapravati 2000, WHO-UNAIDS 
  Report 2001).  
Indeed, the first Brazilian Phase 
  I vaccine trial was conducted in Brazil in 1994, testing an HIV-1 subtype B 
  gp120 V3 loop peptide-based vaccine concept (MN-V3) in 30 healthy volunteers 
  from two cities (Rio de Janeiro and Belo Horizonte). As already described, this 
  vaccine candidate was shown to be safe, but induced a limited antibody and T 
  CD4+ lymphocyte proliferative response to the MN strain vaccine peptide (Sutmoller 
  et al. 1998). More recently, a new HIV-1 subtype B multi-antigen recombinant 
  canarypox/recombinant gp120 Phase I prime-boost protocol was implemented and 
  is presently being conducted in our country.  
FINAL REMARKS  
The results of the first Phase III 
  human trial of HIV-1 vaccines will be available soon and other vaccine concepts 
  like recombinant canarypox/recombinant gp120 prime-boost strategies are planned 
  to enter in the Phase III efficacy evaluation in a near future (Cohen 2001) 
  in some world regions, including South America (WHO-UNAIDS Report 2001).  
Although the immunological relevance 
  of the genetic subtypes for HIV-1 vaccine efficacy is still a matter of extensive 
  investigation and discussion, some international efforts were made for the inclusion 
  of local prevalent subtypes and variant viruses in the vaccine protocols to 
  be tested. For instance, the recombinant gp 120 Phase III trial in USA includes 
  two subtype B variants, MN and a subtype B primary isolate, whereas the protocol 
  being conducted in Thailand employs subtype B MN and a region-specific gp120 
  subtype E primary isolate (Mulligan & Weber 1999). In the African continent, 
  where several HIV-1 subtypes and recombinant viruses predominate, a subtype 
  A based Phase I protocol is being tested in Kenya (Mwau et al. 2001). Considering 
  the spreading of subtype C worldwide, comparative analysis of subtype C samples 
  from different countries, including Brazil, are being performed with the objective 
  of future vaccine development (Rodenburg et al. 2001).  
In conclusion, the development of 
  safe HIV-1 vaccines able to induce broad and long-lasting immune responses affording 
  protection against the array of prevalent variants already described worldwide 
  (including subtypes, recombinants and variant viruses) is presently a distant 
  target to be achieved. However, even considering the difficult challenges to 
  be overcome, urgent vaccine measures are of high priority for public health 
  in order to control the devastating spread of HIV mainly in the poorer world 
  areas. Based on the knowledge already accumulated over time, it is important 
  to stimulate Phase I/II trials for the scientifically defined promising vaccine 
  concepts as well as to move on to Phase III with the more immunogenic and feasible 
  ones. It is of paramount importance to stimulate developing countries to participate 
  in the process of vaccine evaluation, not only testing vaccines according to 
  already defined protocols. But, working together with them, in order take into 
  consideration their local information on virus diversity and host genetic background 
  relevant for the vaccine development and testing, as well as including local 
  virus based reagents to evaluate the immunogenicity of the candidate vaccines 
  is of utmost importance as well.  
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