AIDS Research and Human Retroviruses

The purpose of this study was to generate data on the relative prevalences of the HIV-1 subtypes circulating in Nigeria. A total of 252 HIV-1-positive samples collected during an epidemiologic survey conducted in April 1996 were genetically characterized by HMA (heteroduplex mobility assay) and/or sequencing. Samples were collected in Lagos, Calabar, Kano, and Maiduguri. Overall, the predominant env subtypes were A (61.3%) and G (37.5%). Subtype A is more prevalent in the south (p < 0.001), about 70% in Lagos and Calabar, whereas a quarter of the samples was classified as subtype G in these states. In contrast, subtype G is predominant in the north (p < 0.001), representing 58% of the samples in Kano. In the northeastern region, Maiduguri, almost similar proportions of subtype A and G were seen, 49 and 47.4%, respectively. A total of 37 samples was also sequenced in the p24 region from the gag gene; 13 (35%) had discordant subtype designations between env and gag. The majority of the gag (12 of 17) and env (14 of 22) subtype A sequences clustered with the A/G-IBNG strain. Within subtype G, three different subclusters were seen among the envelope sequences. These different subclusters are observed among samples obtained from asymptomatic individuals and AIDS patients from the four Nigerian states studied. In conclusion, we observed a limited number of HIV1 subtypes circulating in Nigeria, with subtypes A and G being the major env subtypes responsible for the HIV-1 epidemic. Nevertheless, the high rate of recombinant viruses (A/G) and the different A/G recombinant structures indicate a complex pattern of HIV-1 viruses circulating in this country.

We studied the phylogeny of HIV-1 subtype F viruses from children and adults in Romania in order to (1) clarify whether the Romanian subtype F epidemic was caused by one or several virus introductions and(2) gain insight into the route of spread of the HIV-1 subtype F virus among children and adults in Romania. env (V3), gag (p17/half p24), and pol (prot/half RT) sequences were obtained from three districts in Romania: Tirgu Mures (n = 9, children), Craiova (n = 15, children), and Bucharest (n = 13, adults). Of 37 HIV V3 sequences from Romania, 35 belonged to the genetic subtype F in the neighbor-joining tree, whereas 2 sequences from adults clustered with subtypes A and C. Within the subtype F cluster, no bootstrap-supported subclusters were observed according to geographic area in Romania. Two of the adult V3 sequences that clustered with the children were obtained from individuals who tested HIV seropositive in 1989 and 1990, showing that the subtype F virus was present among adults when the HIV epidemic began among children in Romania. The HIV-1 subtype F viruses obtained from children showed a mean pairwise V3 nucleotide distance of 7.9% and maximum distances of between 18 and 19%; both are higher than previously described. The mean V3 distances (overall, synonymous, and nonsynonymous) were significantly higher for adults than for children. One V3 sequence from the Democratic Republic of Congo clustered within the Romanian sequences, suggesting that the subtype F virus in Romania may originate from this area. Our data also suggest that HIV1 subtype F was present among Romanian adults before it appeared in 1989 among institutionalized children. The juvenile population was most likely infected with the HIV-1 subtype F virus on more than one occasion, presumably through HIV-contaminated blood (products) obtained from adults.

A 32-amino acid HIV-1 Gag immunogen was assessed for its ability to augment existing virus-specific CTL responses in chronically HIV-1-infected individuals. The immunogen was an HIV-1 synthetic lipopeptide conjugate composed of an N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl-N-(R)-cysteinyl] group covalently coupled to a synthetic 32-amino acid Gag peptide containing at least 5 CTL epitopes known to be restricted by HLA-A33, -B8, -B27, -B35, and -Bw62. This potential immunotherapeutic was first determined to be safe in six HIV-1-seropositive subjects, with no adverse clinical effects noted during a 182-day period after administration of a dose of 350 mug. The immunogenicity of this lipopeptide conjugate was then assessed in a pilot study in nine HIV-1-seropositive volunteers with peripheral blood CD4⁺ lymphocyte counts of >500/ mul. Three groups of individuals were studied: HLA-selected subjects who received 350 mu g of the immunogen on days 0, 28, and 56 (four subjects); HLA-selected subjects who received a placebo according to a similar inoculation schedule (three subjects); and HLA-mismatched subjects who received the experimental immunogen (two subjects). All subjects were monitored for 26 weeks. After treatment, PBLs from two of the four HLA-selected subjects who received the experimental immunogen showed a transient increase in Gag peptide-specific bulk CTL activity. None of the placebo-vaccinated or vaccinated HLA-mismatched subjects showed any change in bulk Gag peptide-specific CTL activity. However, no consistent decrease in plasma HIV-1 RNA levels was noted in any of the subjects. The present study illustrates that this peptide formulation may not be a sufficiently potent immunogen to significantly augment HIV-1-specific CTLs and to decrease virus load in HIV-1-seropositive individuals.

In Japan, the proportion of hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) is 40%, whereas more than 90% are infected with hepatitis C virus (HCV). To evaluate the immunological status of hemophiliacs infected with HIV-1, we investigated the pattern of cytokine production in peripheral blood mononuclear cells (PBM Cs) of HIV-1-seropositive and-seronegative hemophiliacs, HIV-1-seropositive nonhemophiliacs, and healthy individuals. The production of IL-18 and IL-1 beta from PBMCs stimulated with Staphylococcus aureus Cowan strain 1 (SAC) in the HIV-1-seropositive hemophiliacs was significantly decreased in comparison with the other groups. On the other hand, IL-12 production in both HIV-1-seropositive groups was significantly lower than in HIV-1-seronegative groups. TNF-alpha and IL-6 production was similar among the four groups. In contrast, plasma levels of TGF-beta1 were increased in HIV-1-seropositive hemophiliacs, HIV-1-seropositive nonhemophiliacs, and HIV-1-seronegative hemophiliacs, with the highest levels being in HIV-1-seropositive hemophiliacs, suggesting that coinfection with HIV-1 and HCV increases the level of plasma TGF-beta in HIV-1-seropositive hemophiliacs. Treatment of PBM Cs from healthy individuals with TGF-beta1 inhibited IL-18 and IL-1 beta production without affecting IL-6, IL-10, or TNF-alpha production. Suppression of the expression of caspase 1 mRNA, which is known to be an IL-1 beta-converting enzyme and which also cleaves the precursor of IL-18, was observed in the SAC-stimulated PBMCs from healthy individuals after treatment with TGF-beta1 and in the SAC-stimulated PBM Cs from HIV-1-seropositive hemophiliacs, suggesting that the decreased production of IL-18 and IL-1 beta in HIV-1-seropositive hemophiliacs may be related to the downregulation of caspase 1 mRNA induced by high levels of TGF-beta1 in plasma.

HIV-1 incorporates a variety of host membrane proteins during budding. We have previously shown that adhesion molecules are acquired by the virus in their activated or functional states. Our studies and those of others indicate that adhesion molecules can have profound effects on virus infectivity and its resistance to neutralization by antiviral antibodies. In this study we have examined the effect on infectivity of immobilization or margination of HIV-1 through acquired integrins LFA-1 and VLA-4 onto nonsusceptible cells and solid-phase adhesion ligands (ICAM-1 and VCAM-1, respectively). LFA-1- and VLA-4-mediated HIV-1 binding was supported by ICAM-1 and VCAM-1 immunoglobulin Fc chimeras, respectively. Integrin-mediated HIV-1 binding was also supported by 293 cells transfected with ICAM-1. In both cases the specificity of binding was confirmed with the appropriate blocking monoclonal antibodies or soluble adhesion ligands. We used a sensitive single-cycle infection assay based on a cell line expressing an LTR-luciferase cDNA construct to compare the infectivity of bound virus with that of free virus. Our results show that the binding of HIV-1 to nonsusceptible cells or immobilized adhesion ligands through acquired integrins can increase its infectivity by as much as two orders of magnitude. These results have implications for in vivo dissemination and transmission of HIV-1 and may also explain the high level of virus replication seen in solid lymphoid organs.

Granzymes are a family of serine proteinases commonly found in the granules of CD8⁺ T cells. In HIV infection, CD8⁺ cells show cytotoxic and noncytotoxic antiviral activities. The latter is mediated, at least in part, by a secreted CD8⁺ cell antiviral factor, CAF. Because of the antiviral nature of CD8⁺ cells, we examined the potential anti-HIV activity of free granzymes that can be found in CD8⁺ cell culture fluids. Pretreatment of CD4⁺ T cells with granzyme A or granzyme B had no effect on their susceptibility to infection with HIV, nor did incubation of the granzymes with HIV virions alter their infectivity. Continuous culture of acutely infected CD4⁺ T cells with granzyme A or B showed no effect on cell viability or the replication of HIV. The findings of this study suggest that free granzymes do not control HIV infection and spread in CD4⁺ T cells.

Pertussis toxin (PTX) has been used as a reagent to identify involvement of the G protein-mediated signal transduction pathway. In this study, we found that PTX enhanced HIV-1 replication in acute infection systems at a high dose (1-10 mug/ml) in vitro. PTX treatment enhanced the infectivity of HIV-1-based pseudovirus enveloped with HIV-1 or amphotropic murine leukemia virus (A-MuLV), but not with vesicular stomatitis virus (VSV). This high dose of PTX treatment did not affect HIV-1 gene expression. These data suggested that the effect was virus envelope dependent and that PTX acted on an early stage of viral infection. Treatment with B-oligomer, a nonenzymatic subunit of PTX, mimicked this enhancing effect of PTX. However, desialylation of viral and cellular surface glycoproteins, which are receptors for B-oligomer, did not affect the augmentation induced by PTX. These results indicate that the enhancement of HIV-1 replication is mediated through an unknown biological function of B-oligomer.

The targeted lymph node (TLN) immunization strategy was investigated in macaques, in order to determine the efficacy in generating secretory, systemic, and cellular immune responses, CD8⁺ T cell-generated suppressor factors, and beta-chemokines. TLN immunization of the rectal and genital mucosa-associated iliac lymph nodes (TILNs) was compared with axillary TLN immunization (TAxLN) using HIV-1 M N/LAI gp140^env and SIV p27^gag in alum. Significantly higher immune responses, as well as CD8⁺ T cell-generated anti-SIV factors and the beta-chemokines RANTES, MIP-1 alpha, and M IP-1 beta, were elicited by iliac as compared with axillary TLN immunization. The immune responses induced by TLN immunization were examined for their capacity to prevent rectal mucosal infection by the pathogenic dual-tropic SHIV-89.6P. Despite significant secretory, serum, cellular, and beta -chemokine responses, the macaques were infected by SHIV-89.6P. Whether the lack of protection was associated with the antigenic unrelatedness of SHIV-89.6P to the immunizing HIV-1 MN/LAI gp140 or to the virus utilizing CXCR4 to a much greater extent than CCR5, remains to be determined.