Analysis of HIV-1 quasispecies sequences generated by High Throughput Sequencing (HTS) using HIVE

The high level of genetic variability of Human Immunodeficiency Virus type 1 (HIV-1) is caused by the low fidelity of its replication machinery. This leads to evolution of swarm-like viral populations often described as quasispecies. High throughput sequencing (HTS) technology provides higher resolution over Sanger sequencing, enabling detection of low frequency variant genomes. However, quasispecies analysis is still a challenge due to the systematic noise, introduced by HTS technology. This leads to the increase in type I errors (also known as false positives) and the underlying genetic diversity, which can lead to mathematically insolvable type II errors (also known as false negatives). We have developed a pipeline using the tools in the High-performance Integrated Virtual Environment (HIVE), an HTS platform designed for big data analysis and management, to analyze viral populations within each sample and identify their subtype classification and recombination patterns of recombinants. RNA was extracted from 70 plasma samples of chronic HIV-1 infected patients. The 3’ half genomes of HIV-1 were amplified using RT-PCR and PCR products were sequenced using Illumina MiSeq. The paired end reads for each sample were assembled using Geneious software and analyzed for presence of HIV-1 quasispecies using HIVE tools. Subtype analysis of 70 samples using Geneious software identified 17 A1s, 4 Bs, 30 Cs, 1 D, 6 CRF02_AG, and 12 unique recombinant forms (URFs). Additionally, we found up to 178 ambiguous bases in the consensus sequences from 41 viral samples (58.6%), suggesting the presence of viral subpopulations. However, Geneious could not determine the major viral populations in each sample. We analyzed the same HTS reads using the HIV-1 quasispecies analysis pipeline and found one predominant population in 11 samples (15.7 %), two to ten distinct populations in 45 samples (64.3%), 11-20 in 13 samples (18.16%), and 26 in one sample (1.4 %). Interestingly, two equally major viral populations that were not detected by Geneious were identified in five samples (7.1%) by HIVE. The HIV-1 quasispecies analysis pipeline is reliable and more sensitive in its ability to identify distinct viral populations and the recombination patterns not identified by the Geneious software

Transmitted/Founder and Chronic Subtype C HIV-1 Use CD4 and CCR5 Receptors with Equal Efficiency and Are Not Inhibited by Blocking the Integrin α4β7

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently

Cross-reactive monoclonal antibodies to multiple HIV-1 subtype and SIVcpz envelope glycoproteins

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross–reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly-reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses

Expression and Immunological Characterization of the Carboxy-Terminal Region of the P1 Adhesin Protein of Mycoplasma pneumoniae

Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia in humans. Adherence of M. pneumoniae to host cells requires several adhesin proteins, such as P1, P30, and P116. A major limitation in developing a specific diagnostic test for M. pneumoniae is the inability to express adhesin proteins in heterologous expression systems due to unusual usage of the UGA stop codon, leading to premature termination of these proteins in Escherichia coli. In the present study, we successfully expressed the C-terminal (P1-C1) and N-terminal (P1-N1) regions of the P1 protein in E. coli. On screening these recombinant proteins with sera from M. pneumoniae-infected patients, only the P1-C1 protein was found to be immunogenic. This protein can be used as an antigen for immunodiagnosis of M. pneumoniae infection, as well as in adherence inhibition studies to understand the pathophysiology of the disease

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals

Additional file 10 of Single-cell analysis of immune cell transcriptome during HIV-1 infection and therapy

Additional file 10. Table S13: Genes correlated or anti-correlated with HIV-1 gag transcript expression

Additional file 9 of Single-cell analysis of immune cell transcriptome during HIV-1 infection and therapy

Additional file 9. Table S12: Genes differentially expressed between HIV-1-treated compared to seronegative CD8+ T cells