AIDS Research and Human Retroviruses

HIV-1 infection in children less than 15 years of age is mainly due to mother-to-child transmission. The aim of this work was to investigate molecular evidence to prove father-to-child horizontal transmission in two possible events of transmission. In the first event a boy was identified as HIV infected at 2–3 years of age. At the same time infection was confirmed in the father, while mother and siblings were negative. In the second event a girl was negative for HIV at age 1 and identified as HIV-1 infected at age 6. The father's HIV infection was diagnosed in the same period while the mother was repeatedly negative. No evidence of sexual assault or transfusion was recorded in any case. Peripheral blood mononuclear cells were obtained from both fathers and children. After PCR amplification, the C2V3 region of the envelope gene and the region coding for amino acid 132 of p24 up to amino acid 40 of p7 of the gag gene were sequenced. Genetic distance measurements and phylogenetic tree analysis showed that in both cases the father's and child's viral sequences were closely related. They were distinct when compared to Argentine sequences including sequences from the same geographic region. Epidemiological and molecular data strongly suggest that horizontal transmission had occurred, probably related to the close father-to-child contact.

Hydroxyurea (HU) has preferential activity in virus reservoirs not effectively targeted by current antiretroviral drug regimens, but concern for potential toxicity has precluded its routine use. The effect of adjunct HU on T cell proliferative responses and phenotypic markers was examined in a randomized study of 39 chronically HIV-1-infected patients with virological suppression on potent antiretroviral therapy. While patients in the HU arm showed modest declines in the median CD4⁺ T cell counts (total, –151 cells/mm³; naive, –91 cells/mm³), no significant differences were noted in the Candida, HIV-1 p24, and HIV-1 gp160 responses between the treatment arms following 24 weeks of therapy. In conclusion, although adjunct HU was associated with modest declines in the CD4⁺ T cell counts, there was no significant adverse effect on helper T cell function. Further trials to address the role of HU in HIV-1 treatment may be appropriate after careful selection of HU dose and the adjunct drugs to avoid nonhematological toxicity.

Lipodystrophy resulting from HIV disease or the antiretroviral therapy used to treat it is a common complication. We report on the treatment of cervical lipohypertrophy ("buffalo hump") with suction-assisted lipectomy in six patients.

Two HIV-1 intersubtype recombinant forms are circulating widely in populations and have become important strains in the pandemic: CRF01_AE in Southeast Asia and CRF02_AG in West and West Central Africa, respectively. Several other circulating recombinant forms (CRF) have also been identified, but with fewer numbers of infections and/or more limited geographic spread. Here we expand knowledge of HIV-1 CRF using clinical samples, principally from West Africa, that were difficult to classify by partial genome sequencing. DNA was extracted from primary patient peripheral blood mononuclear cells (PBMC). The virtually complete HIV-1 genome was amplified by polymerase chain reaction (PCR) and directly sequenced. Additional strains were characterized by partial envelope sequencing. Phylogenetic analysis was used to identify and map intersubtype recombination breakpoints. Four virtually complete genome sequences and two partial envelope sequences represent CRF09_cpx, a newly identified complex recombinant HIV-1 whose principal focus seems to be in West Africa. This recombinant includes segments of subtypes A, F, G, and unclassified genetic material. It shares unique unclassified regions with the early Zaire strain Z321. There are similarities in structure, but considerable genetic distances, between CRF09_cpx and CRF02_AG IbNG. In conclusion, it is possible that this CRF shared common ancestors with both Z321 and CRF02_AG in the course of the pandemic, perhaps arising by recombination between earlier forms of these strains. Although newly identified, at least one infection with CRF09_cpx has already occurred outside of Africa.

Highly active antiretroviral therapy (HAART) is associated with a dramatic clinical benefit to HIV-infected patients through significant plasma viremia reduction and CD4⁺ T cells increase. In previous reports, HIV-specific CD4⁺ and/or CD8⁺ T cell responses have been studied separately during HAART; therefore the relationship between these two virus-specific populations is currently not well understood. In this study, both HIV-specific CD4⁺ and CD8⁺ T cell responses were investigated using a large panel of well-defined T cell epitope peptides in 24 HIV-1-infected patients undergoing HAART, with undetectable viral load and CD4⁺ T cell count ≥ 350/mm³. One-third of the patients had CD4⁺ T cells able to proliferate when exposed to HIV-1 protein fragments but only two patients displayed polyclonal responses. In addition the majority (78%) of HAART-treated patients displayed no or monospecific CD8⁺ T cell responses and the phenotypic analysis of these HIV-specific CD8⁺ T cells demonstrated the absence of terminally differentiated effectors. In conclusion, the experimental approach used in this study shows that CD4⁺ T cell responses may persist during HAART but are not associated with strong CD8⁺ T cell responses

Synthetic peptides of sequences within the HIV-1 gp41 heptad repeat-regions (HR-1 and HR-2) can effectively inhibit cell fusion and viral entry. DP178 (T-20), an HR-2 peptide, acts by inhibiting the association between HR-1 and HR-2, thereby interfering with HIV-1 fusion and viral entry. HR-2 peptide binding is predicted to be an important indicator of the presence of Env gp41 fusion intermediate conformation. A stabilized HR-2/Env conjugate might be an HIV-1 vaccine candidate and have the potential for inducing antibodies against transiently exposed epitopes on HIV-1 Env. To explore the possibility of design of HR-2 stabilized-HIV-1 immunogens, we studied the ability of HIV-1 Env to bind to HR-2 peptides. Using surface plasmon resonance (SPR)-binding assays and precipitation of soluble Env gp120 proteins with HR-2 peptide DP178, we have found that there is an HR-2 peptide-binding site on soluble HIV-1 recombinant gp120. Binding of DP178 was induced by sCD4 and by the anti-gp120 human mAb A32. The induction of DP178 binding was inhibited > 80% by the HIV-1 coreceptor-binding site mAb 17b. Binding of DP178 to gp120 was also inhibited by gp120 C4 peptides with sequences that are centrally located within the HIV-1 coreceptor-binding site. Thus, in addition to interactions with the gp41 HR-1 region, the fusion inhibitor peptide DP178 binds to triggered soluble HIV-1 recombinant gp120 following its interaction with sCD4 or CD4 mimic mAb A32. This may prove to be an important consideration when designing an HIV vaccine that utilizes constrained HIV Env proteins.

We explored the use of a simian–human immunodeficiency virus (SHIV) DNA vaccine as an effective mucosal priming agent to stimulate a protective immune response for AIDS prevention. Rhesus macaques were vaccinated rectally with a DNA construct producing replication-defective SHIV particles, and boosted with either the same DNA construct or recombinant modified vaccinia virus Ankara (MVA) expressing SIV Gag, SIV Pol, and HIV Env (MVA-SHIV). Virus-specific mucosal and systemic humoral and cell-mediated immune responses could be stimulated by this approach but were present inconsistently among the vaccinated animals. Rectal vaccination with either SHIV DNA alone or SHIV DNA followed by MVA-SHIV induced SIV Gag/Pol- or HIV gp120-specific IgA in rectal secretions of four of seven animals. However, the gp120-specific rectal IgA antibody responses were not durable and had become undetectable in all but one animal shortly before rectal challenge with pathogenic SHIV 89.6P. Only the macaques primed with SHIV DNA and boosted with MVA-SHIV demonstrated SHIV-specific IgG in plasma. In addition, these animals developed more consistent antiviral cell-mediated responses and had better preservation of CD4 T cells following challenge with SHIV 89.6P. Our study demonstrates the utility of a rectal DNA/MVA vaccination protocol for the induction of diverse responses in different immunological compartments. In addition, the immunity achieved with this mucosal vaccination regimen is sufficient to delay progression to AIDS.

The aim of this study was to evaluate the immunological responses induced by DNA plasmids containing HIV regulatory genes administered in combination in HIV-1-infected patients with pretreatment with highly active antiretroviral treatment (HAART). The study is a double-blind, randomized, and placebo-controlled study, including 15 asymptomatic HIV-1-infected patients on stable HAART for at least 6 months and with plasma HIV RNA levels below 50 copies/ml. Ten patients received a combination of rev, tat, and nef intramuscularly (im) at weeks 0, 4, and 16 at increasing doses giving totals of 300 (100 × 3), 900 (300 × 3), and 1800 (600 × 3) μg DNA. Five patients received saline in the same amounts im. Antigen-specific cytotoxic T lymphocyte (CTL) levels were preserved or increased and new T lymphocyte proliferative responses were induced in the group immunized with the HIV DNA genes. No increase in antibody levels was noted. Despite a 10-fold higher vaccine dose, patients on HAART did not respond better to vaccination compared to non-HAART patients included in a previous study where the genes were administered separately. Combining the regulatory genes rev, tat, and nef in increasing doses may reduce the anticipated augmentation of HIV-specific T cell proliferative and CTL responses. Viral suppression did not seem to further improve the initial vaccine responses of patients with comparable CD4 levels.

Dendritic cells (DCs) infected with recombinant avipox vectors express the introduced genes and activate antigen-specific T cells. DCs exhibit distinct differentiation-dependent immune functions. Moreover, immature DCs are readily infected by canarypox vectors, but undergo tumor necrosis factor (TNF)-α-dependent death, while fewer mature DCs get infected and resist dying. A pilot study was performed using the rhesus macaque system to explore whether immature and mature DCs infected with SIV-recombinant canarypox (vCP180) ex vivo could induce primary virus-specific immune responses in vivo. After subcutaneous (sc) reinjection, functional monocyte-derived DCs migrated to lymph nodes (LNs) within 1–2 days and primed T cells in vivo. This was observed by monitoring dye-labeled DCs in the draining LNs and tetanus toxoid (TT)-specific T cell responses after injection of TT-loaded DCs. DCs from simian immunodeficiency virus (SIV)-naïve rhesus macaques were infected with vCP180 (SIVmac142 gag, pol, and env genes), and sc reinjected into donor animals. Low-level SIV-specific T cell proliferation, but little if any interferon (IFN)-γ production was detected. DCs pulsed with vCP180 in combination with TT and keyhole limpet hemocyanin (KLH) (to activate additional T cells and provide "helper" cytokines) induced SIV-, TT-, and KLH-specific T cell responses, including IFN-γ responses not seen when vCP180-carrying DCs were used alone. Interleukin (IL)-10 and low-level antibody responses were also observed. This pilot study provides the proof of principle that sc injected ex vivo SIV-recombinant canarypox-infected DCs safely induce low-level SIV-specific immune responses in vivo .

To monitor HIV-1 diversity in Argentina, a phylogenetic-based analysis of HIV-1 partial pol sequences obtained for resistance testing in 587 treatment failure patients was performed in Buenos Aires city between 2001 and 2003. HIV-1 RNA was isolated from plasma samples and partialpol fragments amplified by RT-PCR. Sequences were obtained by automated sequencing. Phylogenetic analysis was performed and recombination patterns characterized. A total of 299 sequences grouped into clade B (50.94%) and 284 were B/F recombinants (48.38%). Four sequences were grouped into clades A, C, and F (0.68%). The clade C sample, 96105, was found to be a BC recombinant and samples 103396 and 104575 showed the same mosaic pattern with Kisii5009 from Kenya and 97KR004 from Korea, previously described as A2D recombinants. With the presence of two full-length genomes, one from Kenya and one from Korea, and now two partial genomes from Argentina, this recombinant is designated CRF16_A2D. Its presence on three continents shows that CRF16_A2D has a global distribution.

A limited number of full-length gp120 sequences are currently available for subtype C HIV-1 from India. Sequence data from HIV-1 subtype C in early seroconverter stage virus are also very limited. With the objective of identifying the sequence variation in early seroconverters, we compared Indian subtype C gp120 sequences obtained from six early seroconverters presented in this study with non-Indian subtype C sequences from other parts of the world obtained from the Los Alamos database and subtype C potential vaccine candidate sequences. All these samples were collected within a few weeks of seroconversion and hence they represent gp120 sequences of currently circulating viral strains in India. The phylogenetic tree indicated that the Indian sequences compared here clustered together within the C clade. The seroconverter sequences presented in the study would surely help in identifying the immunogenic epitopes and could be utilized further for developing effective prophylactic strategies against HIV-1 subtype C for India.

HIV-1 is endemic in Tanzania where three different subtypes, A, C, and D, have been identified. Information on HIV-1 genetic diversity is crucial to define requirements for an effective vaccine, in regions where HIV-1 vaccine trials are planned. To define the subtype distribution of HIV-1 in the Mbeya region of southwest Tanzania, peripheral blood mononuclear cells (PBMC) and plasma were obtained from 36 discarded HIV seropositive blood units. Multiregion hybridization assay (MHA) was performed on both PBMC DNA and plasma RNA to determine the subtype distribution. Twenty virtually full-length HIV-1 sequences were amplified from the extracted DNA, sequenced, and phylogenetically analyzed. Subtype distribution determined by all three assays was comparable. More than 50% of the samples analyzed were subtype C, followed by a high proportion of subtype C-containing intersubtype recombinants. Based on this work, subtype C appears to be the prevalent subtype in southwest Tanzania, followed by a high proportion of intersubtype recombinants.

HIV-infected plasma specimens, collected in Cameroon between 1999 and 2002, were screened for HIV-1 group N and SIVcpz infections using a serological screening algorithm based on immunoassays with antigens derived from HIV-1 group M, N, and O, and SIVcpz strains. Specimens with reactivity to group N and SIVcpz antigens were characterized by RT-PCR and sequence analysis to identify the infecting virus. Although several specimens were serotyped as potential group N or SIVcpz infections, only one group N infection was confirmed. The specimen, 02CM-DJO0131, was collected in 2002 from a hospital patient at the D'Joungolo Hospital, Yaoundé. The virus genome was amplified as seven overlapping fragments comprising 8938 nucleotides. Phylogenetic analysis shows that 02CM-DJO0131 branches with group N sequences. With this study, three near full-length sequences are now available for group N. While we confirm the presence of group N in the Cameroonian population, group N infections continue to be rare and difficult to identify.