Reference: Development and Applications |
of Vaccines and Gene Therapy in AIDS
(International Workshop, Naples, June 15-16, 1995).
Editors: G. Giraldo, D.P. Bolognesi,
M. Salvatore, E. Beth-Giraldo.
Vol 48 Antibiotics and Chemotherapy
(Editor: H. Schonfeld).
Karger, Basel, 1996, pp 30-38.
Saladin Osmanov, William L. Heyward, José Esparza
Vaccine Development Unit,
Division of Research and Intervention Development,
Global Program on AIDS, World Health Organization,
More than 18 million human immunodeficiency virus (HIV) infections are estimated to have occurred worldwide since the beginning of the epidemic . If the present trend continues, by the year 2000 there will be a cumulative total of 30 to 40 million HIV infections in the world, with 90% of these infections occurring in developing countries. A safe, effective and affordable HIV preventive vaccine may be essential for the future control of this pandemic [2,3].
However, the development of such a vaccine will be a long and difficult process. Perhaps the two major scientific challenges for HIV vaccine development are the lack of information on immune responses known to correlate with protection against HIV infection, and the extensive genetic variability which is characteristic of this virus [4-7]. These two problems are interrelated. On the one hand, the lack of information on immune correlates of protection limits our ability to rationally design candidate vaccines based on a judicious selection of relevant B- and/or T-cell epitopes present in variable and/or conserved regions of the different viral proteins. Nevertheless, we do not know what could be the biological and antigenic significance of the observed HIV genetic variability. However, such variability has been generally perceived as a major obstacle to the development of broadly effective vaccines.
The acquired immunodeficiency syndrome (AIDS) is caused by two different human immunodeficiency viruses, HIV-1 and HIV-2, which belong to the lentivirus family of retroviruses. Another large group of related lentiviruses has been discovered in nonhuman primates, and designated as simian immunodeficiency viruses (SIV). All known primate lentiviruses are grouped into five distinct phylogenetic lineages. One of these lineages includes all HIV-1 isolates, as well as viruses from chimpanzees (SIVCPZ). A second lineage includes multiple strains of HIV-2, and viruses isolated from sooty mangabeys (SIVMN) and from captive macaques (SIVMAC). The other three lineages include viruses isolated from different species of African monkeys [7-9].
Phylogenetic analyses of the nucleotide sequences of the envelope (env) and core (gag) genes of a large number of HIV-1 isolates, have identified the existence of at least eight different genetic subtypes (clades) of HIV-1, which have been designated A through H [10-16]. These genetic subtypes, which form the major group of HIV-1 (group M), are approximately equidistant to each other, although subtypes B and D seem to be more closely related. A second group of HIV-1 isolates, distantly related to group M viruses, has been recently identified in patients from Cameroon, and designated as group O viruses (for "outliers"). This group of viruses is very heterogeneous, and in fact it may comprise a number of distinct genetic subtypes, which remain to be identified and characterized [17,18].
It is important to indicate, however, that these genetic subtypes, or clades, are not necessarily equivalent to antigenic or immunological subtypes, and that at the present time it is not known what could be their relevance to vaccine-induced protection.
The extensive genetic variability of HIV is primarily due to the high error rates of the viral reverse transcriptase, which results in approximately 10 genomic base changes per replication cycle. In addition to substitutions, deletions and insertions also occur, although the frequency of these genetic errors is more difficult to estimate. The envelope gene (env) seems to be subject to the most extensive genetic variation, although alterations also occur in other genes [7,8,11-13]. HIV-1 undergoes continuous genetic variability within individual patients, who usually harbour a swarm of highly related but individually distinguishable viral variants, which are referred to as quasispecies, with a heterogeneity usually not exceeding 2-5% in the env gene [19,20].
On the other hand, a broad range of viral genetic variability (in the range of 20-30% in the env gene) has been documented for isolates from distinct geographical locations. This phenomenon is largely attributable to differential geographic distribution of the multiple genetic subtypes of HIV-1. Within a single geographic region, the range of genetic variability in the env gene is estimated to be 6-19%, although differences higher than 30% have been documented [8-13,21,24]. The extent of genetic variability in a given geographical location increases over time after the introduction of a particular subtype in a population. Initially, the heterogeneity in the env gene can be as low as 3-5%, which is comparable with the range of intra-patient variability, with further diversification at an estimated rate of approximately 1% per year. If immune protection is dependent on variable HIV sequences, then HIV vaccine development will become a moving target, which may require the continuous adaptation of specific vaccines, as is the case today with influenza vaccines.
In addition, recent data have provided evidence that genomic recombination between two different HIV-1 populations frequently occurs in vivo, resulting in biologically viable viruses with mosaic genomes, a phenomenon which may result in additional HIV genetic variability and viral genetic shifts [25-28]. The identification of these recombinant viruses indicates that co-infection, or super-infection, with different genetic variants of HIV-1 does happen in nature, and this may suggest that active infection with one virus strain does not necessarily confer complete protection against infection with another strain. However, experiments with live-attenuated SIV vaccines in macaques suggest that protection against super-infection is achieved only after an extended period during which protective mechanisms, involving either immune responses or target cell resistance due to interference, are developed in response to the initial virus infection, and have reached the level required for protection.
HIV-1 genetic subtypes are unevenly distributed in different geographical locations [8-13,21-24]. Subtype B viruses are more prevalent in North America, Latin America and the Caribbean, Europe, Japan and Australia. Almost every subtype is present in sub-Saharan Africa, with subtypes A and D predominating in central and eastern Africa, and subtype C in southern Africa. Subtype C is also prevalent in India and it has been recently identified in southern Brazil . Subtype E was initially identified in Thailand, and is also present in the Central African Republic [10,13,22-24]. Subtype F was initially described in Brazil and in Romania . The most recent subtypes described are G, found in Russia and Gabon, and subtype H, found in Zaire and in Cameroon [15,16]. As mentioned before, group O viruses have been identified in Cameroon and also in Gabon [17,18].
However, the above information on the geographical distribution of HIV-1 genetic subtypes is mostly based on limited data which has been generated from small descriptive studies. Well-designed molecular epidemiology studies are thus needed to obtain statistically significant information on the distribution of HIV-1 subtypes in different parts of the world, and to establish possible correlations between genetic subtypes and biological and/or epidemiological characteristics, including mode of transmission and possible segregation of specific HIV-1 subtypes in particular populations with different risks for HIV-1 infection . Studies are also required to better understand the dynamics and driving forces of the epidemic, including cumulative prevalence, as well as incidence of infection with different subtypes. This may allow tracking the spread of the epidemic between different populations, countries, and continents. Such information will be required for the appropriate design of efficacy trials of HIV candidate vaccines, and may be essential for planning strategies for the utilization of future HIV/AIDS vaccines.
The practical implementation of these molecular epidemiology studies is being greatly facilitated by the development of two simple techniques for HIV-1 subtyping, the heteroduplex mobility assay (HMA) and V3-peptide ELISA. Since HMA relies on the amplification of proviral DNA, an important limitation of the technique has been the need to obtain peripheral blood mononuclear cells from infected individuals [20,21,30]. However, HMA is now being modified by the introduction of a reverse transcription step, which will allow the use of small amounts of serum or plasma as a source of viral sequences . The other technique for HIV subtype determination is based on ELISA reactivity of patient's serum with peptides derived from different V3 loop sequences. This is a very practical approach for large-scale screening and is particularly useful in locations where only a few well characterized subtypes are known to be present. Considerable serological cross-reactivity has been observed between A and C subtypes, suggesting that these two genetic variants may fall within a single serotype. Cross-reactivity has also been observed between subtype B and subtype D sera, although to a lesser extent. The genetic variation observed in the V3 region of subtype B viruses from different geographical locations (e.g., North America/Europe, Brazil and Thailand) is also reflected in the reactivity in V3 peptide serology, and may identify these viruses as distinct serotypes [32,33]. Different algorithms for HIV subtype determination, combining initial screening by peptide-ELISA followed by HMA, are presently being developed [24,34].
One of the key questions on HIV vaccine development is related to the immunological significance of the observed HIV genetic variability. Do different HIV-1 subtypes represent different immunological subtypes (or immunotypes)? If this is the case, what would be the relevance of these immunotypes for vaccine-induced protection?
One way to approach this problem is by studying the ability of different polyclonal and monoclonal antibodies to bind to viral proteins derived from different HIV-1 subtypes. The genetic variability of the V3 loop regions from different HIV-1 subtypes is reflected in their ability to bind specific antibodies in peptide-ELISA and, as discussed before, this has been exploited for subtyping purposes [32-34]. If neutralizing antibodies directed against the V3 loop sequences are important for protection, then this would impose the need to develop candidate vaccines containing cocktails of multiple V3 sequences representing the observed inter-clade, as well as intra-clade variability in this region.
Strain-specific neutralizing antibodies are generally directed against linear epitopes present in the V3 loop. However, more broadly reactive neutralizing antibodies can be elicited either to other linear epitopes (eg. in gp41) or to conformational epitopes related to the gp120/CD4-binding site [4,5]. It is interesting that monoclonal antibodies against conformational epitopes of subtype B gp120 molecules, were also capable of binding to gp120 molecules from strains belonging to clades A through F, although clade E strains were the least related to clade B viruses [35,36]. Moreover, monoclonal antibodies are also capable of cross-neutralizing primary isolates belonging to different genetic subtypes .
Preliminary checkerboard neutralization assays of HIV-1 primary isolates from different genetic subtypes, using autologous and heterologous plasma, failed to show any defined pattern of genetic subtype-specific neutralization. Most antisera either failed to neutralize primary isolates, or were broadly cross-neutralizing. These studies suggest that genetic subtypes of HIV-1 do not represent classical neutralization serotypes. On the other hand, these findings cannot exclude the possible existence of HIV neutralization serotypes, although they may not correspond to the known genetic subtypes [38,39]. Moreover, cross-neutralization between group M and group O viruses has also been described .
These results are very encouraging, because they indicate extensive antigenic conservation among HIV-1 strains from different subtypes. If neutralizing antibodies are important for vaccine-induced protection, then a broadly effective vaccine may be a real possibility.
The genetic variability of HIV-1 strains has also been correlated with in vitro growth characteristics of the virus. Syncytia-inducing (SI) viruses can replicate in immortalized T-cell lines, inducing syncytia of the target cells. Non-syncytia-inducing (NSI) viruses are unable to replicate in immortalized T-cell lines, but grow in primary CD4+ cells (T lymphocytes and macrophages) [7,41-47]. The biological properties of HIV-1 are largely determined by genetic variation in the env gene, in particular in the V3 loop. Point mutations which lead to substitutions by positively charged amino acids at specific positions in the V3 loop, and which result in an overall increase of its positive charge, strongly correlate with SI properties of the virus [43,44]. In addition, other regions in the env gene, including V1, V2, C4, and the gp41 transmembrane region are also being investigated as potential genetic determinants of the viral biological properties [48,49]. Interestingly, despite substantial genetic variation between different subtypes, the genetic determinants of biologically important domains are largely conserved among the known subtypes .
Although mixtures of SI and NSI virus variants are found at any point in time within a single infected individual, the earliest virus population observed following HIV infection is generally of the NSI phenotype. However, very early in infection, before seroconversion, a selective amplification of SI variants may occur, although it is rapidly controlled and maintained at relatively low levels during the asymptomatic course of the infection. However, the late stages of infection are characterized by the emergence of SI variants, which then take over the NSI virus population [45,50].
Of potential importance for vaccine development is the observation that NSI and SI viruses differ in their susceptibility to neutralizing antibodies, probably due to a different configuration of their outer envelope glycoprotein. More specifically, the V3 loop seems to be relatively inaccessible to V3-specific neutralizing antibodies among NSI isolates, whereas it is more accessible among SI strains . The present generation of HIV candidate vaccines was developed based on SI viruses, and in immunized animals or human volunteers, the neutralizing antibodies that they induced are mostly directed against V3 loop linear determinants. This could explain the reported failure of these candidate vaccines in inducing neutralizing antibodies against NSI "clinical" isolates of the virus. This led to speculation that effective HIV vaccines must induce antibodies capable of neutralizing "clinical" isolates, perhaps by inducing antibodies directed against conformational epitopes present in multimeric gp120 molecules. However, chimpanzees immunized with monomeric gp120/MN candidate vaccines have been protected from a challenge with a closely related "clinical" strain (SF2), even when the sera from the immunized animals failed to neutralize the virus (D. Francis, personal communication). It remains an open question if protection can be equally achieved against SI and NSI viruses.
The Global Program on AIDS of the World Health Organization (WHO) established in 1992 the "WHO Network for HIV Isolation and Characterization", to promote the development of appropriate HIV vaccines for worldwide use, including in developing countries. The WHO Network has the following major objectives: (1) to standardize and validate different methodologies for HIV isolation and characterization, (2) to monitor HIV variability on a global basis, (3) to obtain detailed genetic and biological characterization of HIV strains, (3) to generate and distribute well-characterized vaccine-related reagents, and (4) to transfer appropriate technologies to, and strengthen the infrastructure of, laboratories at the WHO-sponsored HIV vaccine evaluation sites. A large international study conducted in the framework of the WHO Network demonstrated the feasibility and importance of effective collaboration between scientists from developed and developing countries, to address the complex scientific challenges posed by HIV variability in relation to the development of effective HIV-1 vaccines [10,50].
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