Frank A.J. Konings

Cross-Clade Neutralizing Activity of Human Anti-V3 Monoclonal Antibodies Derived from the Cells of Individuals Infected with Non-B Clades of Human Immunodeficiency Virus Type 1

ABSTRACT The majority of global human immunodeficiency virus infections are caused by viruses characterized by a GPGQ motif at the tip of the V3 loop. Characterization of anti-V3 monoclonal antibodies (MAbs) that neutralize isolates with the GPGQ V3 motif is an important step in designing vaccines that will induce such Abs. Consequently, seven human anti-V3 MAbs derived from the cells of individuals infected with non-B-subtype viruses (anti-V3non-B MAbs) were generated from the cells of individuals from Africa infected with circulating recombinant forms CRF02_AG, CRF09_cpx, and CRF13_cpx, each of which contains a subtype A env gene. Sequence analysis of plasma viruses revealed a GPGQ motif at the apex of the V3 loop from six of the seven subjects and a GPGR motif from one subject. The MAbs were selected with fusion proteins (FP) containing V392UG037.8 or V3JR-CSF from subtype A or B, respectively. In virus binding assays, five of the seven (71%) anti-V3non-B MAbs bound to V3-FPs from both subtype A and subtype B, while only four of the nine (44%) anti-V3B MAbs recognized both V3-FPs. Using two neutralization assays, both the anti-V3non-B and the anti-V3B MAbs neutralized subtype B viruses with similar activities, while the anti-V3non-B MAbs exhibited a tendency toward both increased potency and breadth of neutralization against non-B viruses compared to anti-V3B MAbs. Statistical significance was not achieved, due in large measure to the sizes of the MAb panels, but the overall pattern of data strongly suggests that viruses with the GPGQ motif at the tip of the V3 loop induce anti-V3 Abs with broader cross-neutralizing activity than do viruses with the GPGR motif.

Genetic and Biological Properties of HIV Type 1 Isolates Prevalent in Villagers of the Cameroon Equatorial Rain Forests and Grass Fields: Further Evidence of Broad HIV Type 1 Genetic Diversity

To understand the evolution of HIV-1, the genetic and biological characteristics of viruses that infect persons living in regions in which the virus has been evolving for several decades must be studied. Thus, we investigated teh genetic subtypes, coreceptor usage, and syncytium-inducing ability of viruses in 47 HIV-1-infected blood samples from individuals living in rural villages in the equatorial rain forest and grass field regions in Cameroon. Heteroduplex mobility analysis (HMA) of gag (part of p24 and p7) and env (C2V5) or sequence and phylogenetic analysis of gag (part of p24 and p7), pol (protease), and env (C2V5), revealed a broad HIV-1 group M genetic diversity. Subtype analysis revealed genetic evidence of seven subtypes (A, C, D, F, G, H, and J) and three circulating recombinant froms (CRFs) (CRF01_AE, CRF02_AG, and CRF11_cpx). Only 15 (32%) of the 47 samples analyzed revealed a concordant subtype in all three genes (gag, pol, and env), while discordant subtypes and CRFs were identified for the remaining 32 (68%) samples. Two patterns of HIV-1 diversity could be discerned in two provinces. While more concordant subtypes in gag, pol, and env genes were identified in villages of South province (10 of 13, 77%), the HIV-1 diversity in the West province was characterized by intersubtype recombinants (63%). Five new intersubtype recombinants were identified including Agag Jpol Genv, Ggag Upol Aenv, AGgag Jpol Aenv, Agag AGpol Henv, and Cgag AGpol AGenv. All of the 40 viruses tested used the R5 coreceptor, of which four also used the X4 coreceptor. Four viruses were able to induce syncytia in MT-2 cells, however, syncytium induction did not correlate with coreceptor usage. This study further reveals the complexity of HIV-1 infection in rural Cameron and points to the future of the global epidemic, which may be characterized by more genetically diverse viruses.

Protease Mutations in HIV-1 Non-B Strains Infecting Drug-Naive Villagers of Cameroon

To describe the presence of protease inhibitor (PI) resistance-associated mutations and subtype distribution in drug-naive villagers of six provinces of Cameroon, we sequenced the protease (PR) gene (297 bp) of 128 viruses. Secondary PI resistance-associated mutations were identified at five sites: L10I/V (16%), K20R (8%), M36I (98%), L63P (13%), and V77I (6%). No primary mutation in the PR was identified. Of the 128 specimens analyzed, subtypes A (11%), C(2%), D (6%), F2 (3%), G (6%), H (0.8%), J (6%), and CRF02_AG (60%) were identified. The mutations identified were not characteristic to any particular subtype. The absence of primary mutations, in addition to the few secondary mutations, gives good perspectives for PI treatment interventions in these rural areas.

Identification and distribution of HIV type 1 genetic diversity and protease inhibitor resistance-associated mutations in Shanghai, P. R. China.

Objective To investigate the genetic diversity of the HIV-1 circulating in Shanghai and to analyze the mutations in the protease (PR) gene associated with resistance to protease inhibitors (PIs). Design The genetic diversity of HIV-1 and PI resistance–associated mutations was studied in 40 Shanghai HIV-1-seropositive treatmentnaive residents. The patients studied were exposed to the infection mainly through contaminated blood products (hemophiliacs) (n = 17) and sexual contacts (n = 19). Samples from 2 injecting drug users (IDUs) and 2 children born to HIV-1 infected mothers were also analyzed. Methods HIV-1 partial gag, pol, and env genes in infected plasma samples were amplified by reverse transcriptase polymerase chain reaction, sequenced, and phylogenetically analyzed. Analysis of PI resistance–associated amino acid substitution in PR was performed. Results HIV - 1 genes in 38 of the 40 plasma samples were successfully amplified and analyzed. Polymerase chain reaction amplification was successful for 16/17 hemophilia patients and 18/19 sexually infected individuals. While all the 16 hemophilia patients infected through contaminated blood products were infected with subtype B′, the 18 patients infected through sexual contact were infected with several subtypes including subtype A (n = 2), B (n = 4), B′ (n = 1), C (n = 2), CRF08_BC (n = 1), CRF01_AE (n = 7), and intersubtype recombinant CRF01_AE/B (n = 1). The 2 IDUs were infected with CRF08_BC and the 2 children born to HIV-1 infected mothers were infected with subtype B′ and CRF01_AE. PI resistance–associated amino acid substitutions were found at 1 codon in primary and 7 codons in secondary regions of the PR gene. Amino acid substitutions were more frequently found in the B/B′ sequences (69%) than in the non-B sequences (31%). Substitutions characteristic with the subtype B/B′ sequences mainly among hemophiliacs included L63P (87%), A71V/T (27%), and V77I (93%) while those that characterized the non-B sequences mainly found among heterosexuals included M36I (69%) and K20R (19%). Conclusion This study reveals the presence of multiple HIV-1 subtypes and recombinants infecting Shanghai residents. The broad HIV-1 diversity is being introduced into this city through heterosexual contacts. This study also reveals that viruses infecting these treatment-naive patients have acquired both primary or secondary mutations in their PR genes. These studies should provide the basis for further epidemiologic surveys of HIV-1 subtypes and set strategies for treatment intervention and vaccine programs.

Infection with HIV type 1 group M non-B subtypes in individuals living in New York City

Objective: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City. Design: From October 1999 through April 2003, HIV-1–seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes. Methods: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV-1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis. Results: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01_AE–like (n = 7; 13%), CRF02_AG–like (n = 19; 34%), an intersubtype recombinant form Ggag/Aenv (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). Conclusion: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States.

V118I Substitution in the Reverse Transcriptase Gene of HIV Type 1 CRF02_AG Strains Infecting Drug-Naive Individuals in Cameroon

We describe the resistance-associated substitutions that are present in the reverse transcriptase (RT) genes of HIV-1 CRF02_AG strains infecting drug-naive villagers of Cameroon. The 11 sequences analyzed were previously subtyped as CRF02_AG in the gag, pro, and env genes, and this work revealed that most (10/11) had a concordant subtype (CRF02_AG) in the pol gene, while one sequence had discordant subtype (A1) in the pol gene. Classification of the CRF02_AG sequences was further confirmed by recombination breakpoint analysis, which revealed a mosaic composition similar to the reference strain IbNG. Analysis of the RT genes for resistance-associated substitutions revealed two sequences containing a V118I substitution. Even though no other resistance-associated substitutions were found, the presence of V118I, which is implicated in resistance to reverse transcriptase inhibitors, in CRF02_AG strains infecting drug-naive individuals should be considered when introducing these antiretrovirals in areas where CRF02_AG is the predominant subtype, such as Cameroon.