Association between virus-specific T-cell responses and plasma viral load in human immunodeficiency virus type 1 subtype C infection.

Virus-specific T-cell immune responses are important in restraint of human immunodeficiency virus type 1 (HIV-1) replication and control of disease. Plasma viral load is a key determinant of disease progression and infectiousness in HIV infection. Although HIV-1 subtype C (HIV-1C) is the predominant virus in the AIDS epidemic worldwide, the relationship between HIV-1C-specific T-cell immune responses and plasma viral load has not been elucidated. In the present study we address (i) the association between the level of plasma viral load and virus-specific immune responses to different HIV-1C proteins and their subregions and (ii) the specifics of correlation between plasma viral load and T-cell responses within the major histocompatibility complex (MHC) class I HLA supertypes. Virus-specific immune responses in the natural course of HIV-1C infection were analyzed in the gamma interferon (IFN-gamma)-enzyme-linked immunospot assay by using synthetic overlapping peptides corresponding to the HIV-1C consensus sequence. For Gag p24, a correlation was seen between better T-cell responses and lower plasma viral load. For Nef, an opposite trend was observed where a higher T-cell response was more likely to be associated with a higher viral load. At the level of the HLA supertypes, a lower viral load was associated with higher T-cell responses to Gag p24 within the HLA A2, A24, B27, and B58 supertypes, in contrast to the absence of such a correlation within the HLA B44 supertype. The present study demonstrated differential correlations (or trends to correlation) in various HIV-1C proteins, suggesting (i) an important role of the HIV-1C Gag p24-specific immune responses in control of viremia and (ii) more rapid viral escape from immune responses to Nef with no restraint of plasma viral load. Correlations between the level of IFN-gamma-secreting T cells and viral load within the MHC class I HLA supertypes should be considered in HIV vaccine design and efficacy trials.

Magnitude and frequency of cytotoxic T-lymphocyte responses: identification of immunodominant regions of human immunodeficiency virus type 1 subtype C.

A systematic analysis of immune responses on a population level is critical for a human immunodeficiency virus type 1 (HIV-1) vaccine design. Our studies in Botswana on (i) molecular analysis of the HIV-1 subtype C (HIV-1C) epidemic, (ii) frequencies of major histocompatibility complex class I HLA types, and (iii) cytotoxic T-lymphocyte (CTL) responses in the course of natural infection allowed us to address HIV-1C-specific immune responses on a population level. We analyzed the magnitude and frequency of the gamma interferon ELISPOT-based CTL responses and translated them into normalized cumulative CTL responses. The introduction of population-based cumulative CTL responses reflected both (i) essentials of the predominant virus circulating locally in Botswana and (ii) specificities of the genetic background of the Botswana population, and it allowed the identification of immunodominant regions across the entire HIV-1C. The most robust and vigorous immune responses were found within the HIV-1C proteins Gag p24, Vpr, Tat, and Nef. In addition, moderately strong responses were scattered across Gag p24, Pol reverse transcriptase and integrase, Vif, Tat, Env gp120 and gp41, and Nef. Assuming that at least some of the immune responses are protective, these identified immunodominant regions could be utilized in designing an HIV vaccine candidate for the population of southern Africa. Targeting multiple immunodominant regions should improve the overall vaccine immunogenicity in the local population and minimize viral escape from immune recognition. Furthermore, the analysis of HIV-1C-specific immune responses on a population level represents a comprehensive systematic approach in HIV vaccine design and should be considered for other HIV-1 subtypes and/or different geographic areas.

Human immunodeficiency virus type 1 subtype C molecular phylogeny: consensus sequence for an AIDS vaccine design?

An evolving dominance of human immunodeficiency virus type 1 subtype C (HIV-1C) in the AIDS epidemic has been associated with a high prevalence of HIV-1C infection in the southern African countries and with an expanding epidemic in India and China. Understanding the molecular phylogeny and genetic diversity of HIV-1C viruses may be important for the design and evaluation of an HIV vaccine for ultimate use in the developing world. In this study we analyzed the phylogenetic relationships (i) between 73 non-recombinant HIV-1C near-full-length genome sequences, including 51 isolates from Botswana; (ii) between HIV-1C consensus sequences that represent different geographic subsets; and (iii) between specific isolates and consensus sequences. Based on the phylogenetic analyses of 73 near-full-length genomes, 16 "lineages" (a term that is used hereafter for discussion purposes and does not imply taxonomic standing) were identified within HIV-1C. The lineages were supported by high bootstrap values in maximum-parsimony and neighbor-joining analyses and were confirmed by the maximum-likelihood method. The nucleotide diversity between the 73 HIV-1C isolates (mean value of 8.93%; range, 2.9 to 11.7%) was significantly higher than the diversity of the samples to the consensus sequence (mean value of 4.86%; range, 3.3 to 7.2%, P < 0.0001). The translated amino acid distances to the consensus sequence were significantly lower than distances between samples within all HIV-1C proteins. The consensus sequences of HIV-1C proteins accompanied by amino acid frequencies were presented (that of Gag is presented in this work; those of Pol, Vif, Vpr, Tat, Rev, Vpu, Env, and Nef are presented elsewhere [http://www.aids.harvard.edu/lab_research/concensus_sequence.htm]). Additionally, in the promoter region three NF-kappa B sites (GGGRNNYYCC) were identified within the consensus sequences of the entire set or any subset of HIV-1C isolates. This study suggests that the consensus sequence approach could overcome the high genetic diversity of HIV-1C and facilitate an AIDS vaccine design, particularly if the assumption that an HIV-1C antigen with a more extensive match to the circulating viruses is likely to be more efficacious is proven in efficacy trials.

Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for AIDS vaccine design.

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.

Identification of most frequent HLA class I antigen specificities in Botswana: relevance for HIV vaccine design.

Since the mid-1990s, southern African countries have been experiencing an expansion of human immunodeficiency virus type 1 (HIV-1) infection caused by HIV-1 subtype C. To facilitate the design of an HLA-based HIV vaccine, we studied the distribution of the HLA class I antigen specificities in Botswana, a southern African country with a high prevalence of HIV infection. Botswana's highly efficient health care system and its central geographical location within southern Africa suggests that it might be an appropriate candidate site for future trials of an HLA-based HIV vaccine. Specificities of HLA class I genes have been investigated in DNA samples obtained from 161 persons of Botswana origin by polymerase chain reaction (PCR) with sequence-specific primers. We identified 4 HLA-A, 7 HLA-B, and 5 HLA-C specificities that were observed at high frequencies in the Botswana population: A30, A02, A23, A68, B58, B72, B42, B8, B18, B44, B45, Cw7, Cw2, Cw17, Cw6, and Cw4. HLA-A30, A02, A23, A68, B58, Cw2, Cw4, Cw6, Cw7, and Cw17 were observed at frequencies of more than 10%. The frequency of HLA-A30 was 27.3%. HLA-B58 (17.9%) was the most frequent generic HLA-B type. Other frequent antigen specificities detected for the HLA-B were B72 (9.6%), B42 (9.3%), B8 (7.4%), B18 (7.4%), B44 (7.4%), and B45 (6.4%). Analysis of haplotype frequencies revealed that haplotypes HLA-A30/HLA-B58 (6.7%), A30/B42 (6.1%), A30/B8 (4.1%), A30/B45 (3.2%), and A23/B58 (2.5%) were the most frequent among two-locus haplotypes. The comparison of HIV-positive patients and noninfected controls for HLA class I specificities confirmed the previously described association of A2/A6802 supertype with resistance to HIV. Our study suggested an increased resistance to HIV infection associated with A68 rather than A2. We also found that the generic HLA-B58 type was associated with increased susceptibility to HIV infection. Our findings suggest that the design of an HLA-based HIV vaccine that includes multiple CTL epitopes restricted by identified common HLA class I specificities might target up to 97.5% of the population in Botswana. The results of this study extend the HLA map to a southern African country that has high rates of HIV and also provide a database for the design of an HLA-based HIV vaccine.

Molecular cloning and biological characterization of full-length HIV-1 subtype C from Botswana.

Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells.

HIV type 1 A/J recombinant with a pronounced pol gene mosaicism.

A nearly full-length genome sequence of a novel HIV-1 A/J recombinant with a complex structure of the pol gene has been analyzed. This virus was isolated in 1998 from a 35-year-old female from Botswana. The virus demonstrated a dual pattern for CXCR4/CCR5 coreceptor utilization. Using short-term enrichment of the donor's PBMCs, the 98BW21 isolate was long-range amplified, cloned, and sequenced. The sequence of the clone 98BW21.17 spanned 9103 bp from the PBS site to the U5 region of the 3' LTR. The phylogenetic relationship of the 98BW21.17 clone to HIV-1 sequences represented by M, N, and O groups and A-K subtypes of the M group was examined across the entire viral genome. The 98BW21.17 clone demonstrated a unique phylogenetic topology clustering within subtype A or subtype J reference sequences. However, the subtype origin of two regions within the pol gene (p51 RT and integrase) could not be identified. Recombination patterns of the 98BW21.17 clone were different from known AGJ/AGIJ-type viruses such as isolates BFP90 and 95ML84. This study demonstrated the existence and replication competence of a new dual-tropic X4/R5 recombinant form of HIV-1 on the subtype J backbone. The nucleotide sequence of the 98BW21.17 clone was submitted to GenBank under accession number AF192135.