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.

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.

Molecular cloning and phylogenetic analysis of human immunodeficiency virus type 1 subtype C: a set of 23 full-length clones from Botswana.

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9. 1% versus 6.5 and 4.3%, respectively; P < 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.

Pattern of gp120 sequence divergence linked to a lack of clinical progression in human immunodeficiency virus type 1 infection.

Differential rates of AIDS development and/or T4 lymphocyte depletion in HIV-1-infected individuals remain unexplained. The hypothesis that qualitative differences in selection pressure in vivo may account for different rates of disease progression was addressed in nine eligible study participants from a cohort of 315 homosexual men who have been followed since 1985. Disproportionately fewer changes in variable regions and more in C3 of gp12O were found to be significantly associated with slower disease progression. Our finding provides the first example to demonstrate that differential selection pressure related to the emergence of HIV-1 variants is associated with long term nonprogression. Candidate vaccines that elicit strong selection pressure on C3 of gp120 are likely to provide better protection than those targeting variable regions.

High levels of anti-HIV-1 envelope antibodies in cerebrospinal fluid as compared to serum from patients with AIDS dementia complex.

The antibody response to the HIV-1 envelope protein has not been well characterized in patients with AIDS dementia complex (ADC). We evaluated the frequency of antibodies against the HIV-1 envelope in cerebrospinal fluid (CSF) and serum from 21 persons with ADC and 10 symptom-free HIV-1-positive subjects using Western immunoblot with reducing and nonreducing buffer and radioimmunoprecipitation (RIP) analysis. RIP analysis revealed anti-envelope antibodies in all sera tested. Higher anti-envelope levels were observed in CSF than in serum of 12 of 21 ADC patients and only 1 of 10 symptom-free subjects (two-sided Fisher exact test, p < 0.05). All persons with moderate to severe ADC had higher anti-envelope levels in CSF than in sera (p < 0.005). CSF anti-gp120 antibodies were not as readily detected by Western blot analysis even under nonreduced conditions, suggesting that they are directed to conformational epitopes. Higher CSF anti-envelope antibodies appear to be more common in patients with ADC than in symptom-free HIV-1-positive subjects. This antibody pattern may serve as a marker for ADC and its progression.

HIV-1 Langerhans' cell tropism associated with heterosexual transmission of HIV.

Heterosexual transmission by vaginal intercourse accounts for most transmission of human immunodeficiency virus-type 1 (HIV-1) in Africa and Asia but is less important in the HIV-1 epidemics of the United States and Western Europe. Epithelial Langerhans' cells (LCs) represent a possible source of initial cell contact for vaginal infection. Fifteen primary isolates of HIV-1 from U.S. homosexuals and 18 HIV-1 isolates from Thailand heterosexuals were evaluated for growth in LCs of U.S. origin. All the viruses from the Thai heterosexuals, which were subtype E, grew more efficiently in the LCs than any of the viruses from the U.S. homosexuals, which are subtype B. These results suggest that LC tropism is associated with the efficiency of heterosexual transmission of HIV.

Dysregulation of interleukin-7 receptor may generate loss of cytotoxic T cell response in human immunodeficiency virus type 1 infection.

Virus-specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV-1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)-7 can affect T cell maturation and differentiation. Here we report on a group of five HIV-1-positive individuals who tested negative for env- and gag-specific CTL activity. When exogenous recombinant human IL-7 was added as a stimulus to the cultures, none (0/5) of the CTL-negative individuals exhibited a CTL response. Individuals that were negative for HIV-1-specific CTL activity were found to lack IL-7 receptor (IL-7R) on CD8+ cells with a comparable reduction on CD4+ cells. Increased shedding of IL-7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of 125I-labeled IL-7 and Scatchard analysis. The lack of IL-7R is probably not due to endogenous IL-7, since it was not detectable in the culture supernatants of the patients studied. HIV-1 proteins may cause down-modulation of IL-7R expression, either by producing an insufficient number of molecules or by rapid decay of IL-7R on T cells. These changes may alter the cells' capability to respond to the IL-7 growth signal, resulting in CTL failure and subsequent mishandling of the virus.

Killing of primary CD4+ T cells by non-syncytium-inducing macrophage-tropic human immunodeficiency virus type 1.

Understanding the mechanism by which human immunodeficiency virus type 1 (HIV-1) kills CD4+ T lymphocytes is important to the development of therapeutic and prophylactic strategies. Recent studies have indicated that, in some cases, progression to AIDS is associated with the appearance of syncytium-inducing, T cell line-tropic HIV-1 variants. Nevertheless, approximately 50% of subjects with AIDS harbor only non-syncytium-inducing, macrophage-tropic (NSI-M) variants of HIV-1. In most asymptomatic patients, NSI-M HIV-1 isolates are the predominant virus type found. We report here that cytopathicity of NSI-M HIV-1 for primary CD4+ T lymphocytes can be directly detected in vitro. The extent of CD4+ T-cell killing was not completely correlated with the rate of viral replication, suggesting that other characteristics of HIV-1 contribute to its cytopathicity. Our findings suggest that: (i) direct killing by NSI-M HIV-1 may contribute to CD4+ T-lymphocyte depletion in vivo, and (ii) the determinants of HIV-1 cytopathicity for CD4+ T lymphocytes and cell tropism or syncytia-forming ability are not necessarily tightly linked.

Evaluation of autoantibodies to brain proteins in patients with AIDS dementia complex.

A high frequency of autoantibodies to brain proteins has been reported in HIV-1-positive patients. However, the specificity of this response has not been characterized. Using homogenized tissue from three normal brains, the presence of autoantibodies to human brain proteins was analyzed in 16 HIV-1-positive patients with AIDS dementia complex (ADC), 10 HIV-1-positive patients without ADC, 10 patients with multiple sclerosis, 10 patients with juvenile rheumatoid arthritis, and 10 normal controls. Although antibodies to various brain proteins were detected in sera from one-third HIV-1-infected individuals with or without ADC, the proteins recognized were different among different brains. Only one ADC patient had consistent seroreactivity to a 50-kDa brain-specific protein. Our results indicate that autoantibodies to brain proteins are infrequently present in patients with ADC.

Molecular mimicry between the human immunodeficiency virus type 1 gp120 V3 loop and human brain proteins.

Immunologically cross-reactive proteins in the human brain that resemble the V3 loop of human immunodeficiency virus type 1 (HIV-1) gp120 have been identified. When several homogenized tissues from normal brains were used, a monoclonal antibody raised against amino acids 308 to 320 of the V3 loop reacted with three prominent human brain proteins (HBP) of 35, 55, and 110 kDa. Among the three, the 55-kDa HBP appears to be specific to the central nervous system. These results indicate that the V3 loop of HIV-1 gp120 shares an epitope with HBP. An immune response to the V3 loop that generates cross-reactive antibodies to cellular proteins may be an autoimmune mechanism by which HIV-1 can damage the central nervous system.

Mutations in the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein impair the incorporation of Env proteins into mature virions.

In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp41). Truncation of 147 amino acids from the carboxyl terminus of gp41 (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp41 generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp41 required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp41 did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp41 also plays a role in other steps of virus replication.

Unusual pattern of antibodies to human T-cell leukemia virus type-I in family members of adult T-cell leukemia patients.

Detection methods for the human T-cell leukemia virus type-I (HTLV-I) for blood screening and diagnosis generally rely on antibody tests that use the structural proteins of HTLV-I as antigen. We have found an unusual pattern of antibody reactivity among people who are at high risk of HTLV infection due to being a family member of an adult T-cell leukemia (ATL) patient: a specific antibody reaction exclusively directed to the HTLV regulatory protein tax, and not to the HTLV-I structural proteins. Sera from 7 of 82 (8.5%) structural antibody-undetectable family members of ATL patients had the anti-tax reactivity. Two seroconverters were observed. One seroconverter a healthy resident of Miyazaki, tested negative for structural antibody, but positive for tax antibody. Two years later she tested positive for both. The other seroconverter, an Israeli hemophiliac, tested negative for both antibodies, but converted to tax antibody-positive/structural antibody-negative. The HTLV-I tax-only antibody profile was also observed in sera sets from two other populations at risk for HTLV infection, human immunodeficiency virus-1-infected patients at the Bronx-Lebanon Hospital in New York and Israeli hemophiliacs. DNA samples from lymphocytes of four individuals with antibody reactivity only to HTLV-I tax were tested in polymerase chain reaction experiments; no HTLV-I or -II DNA was detected.

The prevalence of antibody to p42 of HTLV-I among ATLL patients in comparison with healthy carriers in Japan.

A gene product (p42) of the long open reading frame, now termed tax, of the viral genome of human T-cell leukemia virus type I (HTLV-I) may be related to the transformation of T cells in adult T-cell leukemia-lymphoma (ATLL). To evaluate its association with the disease, we compared the prevalence of antibody to p42 in sera obtained from 105 HTLV-I carriers and 64 ATLL patients from southwest Japan. The prevalence of the anti-p42 antibody reactivity was 63% among carriers and 31% among cases. The cases were more than 3 times as likely to lack antibody to p42 than carriers, the relative odds (OR) = 3.4, p = 0.001. When the samples were tested for antibody against p24, the most immunogenic core protein, the prevalence was somewhat higher among carriers (65%) than in cases (52%), but not significantly so (p = 0.15). Among the healthy carriers, the correlation between the prevalence of both antibodies was high (p = 0.001), and only 25% of those who had antibody to p24 lacked antibody to p42. However, among the cases, reactivity to both antigens was independent (p = 0.52), and 65% of those with antibody to p24 lacked antibody to p42, OR = 6.3, p = 0.0004. Thus the strongest serologic marker of ATLL following diagnosis was lack of reactivity to p42, particularly among those subjects with anti-p24. Whether this altered response is present prior to disease remains to be determined.

Antibody responses in early human immunodeficiency virus type 1 infection in hemophiliacs.

Sera from hemophiliacs were analyzed for antibodies to human immunodeficiency virus type 1 (HIV-1) by using radioimmunoprecipitation (RIP), western blotting (WB) with nonreducing buffer (NR), and WB with reducing buffer (R). We analyzed envelope gp160, gp120, and gp41; pol gene proteins p64, p53, and p34; and gag gene protein p24. Of 215 samples positive for reactivity to gp160 and gp120(RIP), antibodies to p24 were undetectable in 2 (0.9%), to gp41 in 9 (4.2%), to the pol antigens in 5 (2.3%), to gp120(NR) in 3 (1.4%), and to gp120(R) in 55 (25.6%). By sequential analysis of samples, antibodies to gp120(NR), gp120(R), p24, gp41, p64/53, and p34 were observed later in the course of infection than were antibodies to gp120(RIP) or gp160. This result suggests caution against reliance on WB as the "gold standard." A significantly higher rate of progression to AIDS-related complex was found for individuals lacking antibodies to gp120(R). It is possible that antigenic domains represented by gp120(R) may play a role in the pathogenesis of HIV-1 infection.