Characterization of an imported HIV-1 A1/A7/G recombinant in China.

BACKGROUND: International migration has accelerated the HIV-1 spread across national borders, gradually reducing the restrictions on the geographical distribution of HIV-1 subtypes. Subtypes A and G are globally recognized as the third and sixth most dominant HIV-1 genotypes, mainly prevalent in Africa, but rarely detected in China. Here we reported an imported HIV-1 recombinant which was composed of sub-subtypes A1 and A7 of subtype A and subtype G genes in a Chinese female. This virus was the first HIV-1 recombinant including A7 genes reported in the world. CASE PRESENTATION: The near full-length genome (NFLG) was obtained from the plasma sample of the female in an HIV-1 molecular epidemiological survey with 853 participants in China. Phylogenetic analyses showed that this NFLG sequence contains three A7 segments, four G segments and one A1 segment with seven breakpoints, and all these segments were closely related to HIV-1 references circulating in Africa. The evidence from epidemiological investigation indicated that this female participant had a more-than-two-years heterosexual contact history with a fixed partner from Nigeria, a country in west Africa, which further supported the results of phylogenetic analyses. By the Bayesian phylogenetic analyses, the times of most recent common ancestors (tMRCA) of the partial pol gene (nt2308-3284, A7 region) and full-length vpr-vpu plus partial env gene (nt5534-6858, G region) were estimated around 1989 and 1984, respectively. CONCLUSIONS: In this study, by using the NFLG sequencing, we identified an imported HIV-1 A1/A7/G recombinant which was estimated to originate around 1980s in Africa and introduced into China with international migration. This study highlighted the complexity of the global HIV-1 epidemic, the necessity of using genome sequences to determine HIV-1 genotypes and the importance of real-time monitoring of HIV-1 infection among international migrants and travelers.

Mutations of Glu560 within HIV-1 Envelope Glycoprotein N-terminal heptad repeat region contribute to resistance to peptide inhibitors of virus entry.

BACKGROUND: Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of gp41 can inhibit HIV-1 infection in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that induces fusion between viral and host cell membranes. Previously, we found that HIV-1 acquired the mutations of Glu560 (E560) in HR1 of envelope (Env) to escape peptide inhibitors. The present study aimed to elucidate the critical role of position 560 in the virus entry and potential resistance mechanisms. RESULTS: The Glu560Lys/Asp/Gly (E560K/D/G) mutations in HR1 of gp41 that are selected under the pressure of N- and C-peptide inhibitors modified its molecular interactions with HR2 to change 6HB stability and peptide inhibitor binding. E560K mutation increased 6HB thermostability and resulted in resistance to N peptide inhibitors, but E560G or E560D as compensatory mutations destabilized the 6HB to reduce inhibitor binding and resulted in increased resistance to C peptide inhibitor, T20. Significantly, the neutralizing activities of all mutants to soluble CD4 and broadly neutralizing antibodies targeting membrane proximal external region, 2F5 and 4E10 were improved, indicating the mutations of E560 could regulate Env conformations through cross interactions with gp120 or gp41. The molecular modeling analysis of E560K/D/G mutants suggested that position 560 might interact with the residues within two potentially flexible topological layer 1 and layer 2 in the gp120 inner domain to apparently affect the CD4 utilization. The E560K/D/G mutations changed its interactions with Gln650 (Q650) in HR2 to contribute to the resistance of peptide inhibitors. CONCLUSIONS: These findings identify the contributions of mutations of E560K/D/G in the highly conserved gp41 and highlight Env's high degree of plasticity for virus entry and inhibitor design.

Identification of a novel HIV-1 second-generation circulating recombinant form (CRF136_0107) among MSM in China.

OBJECTIVE: To investigate the molecular epidemiology of HIV-1 in Heilongjiang, China, and try to spot signs of new circulating recombinant form (CRF) in this region. DESIGN: A molecular epidemiological study was conducted in Heilongjiang, China during 2011-2020. METHODS: Plasma samples were collected from three HIV-1-positive patients (two MSM and one man lacking risk factor information). The near full-length genome sequences (NFLGs) of a novel CRF were then obtained and subjected to phylogenetic analysis using Mega 7.0.26. Recombination analysis was performed by the jumping profile Hidden Markov Model (jpHMM). Finally, the origin time of this novel CRF was inferred using the Bayesian phylogenetic analysis in Beast v1.10.4. RESULTS: The three NFLGs formed a distinct monophyletic cluster in the neighbor-joining (NJ) tree. Recombination analysis revealed that the recombinant genome was composed of five segments derived from CRF01_AE, subtypes B, and C, but further confirmed to be a second-generation recombinant form of CRF01_AE/CRF07_BC by a comparison of genome maps and subregion phylogenetic analysis and, therefore, designated as CRF136_0107. With Bayesian phylogenetic analysis, CRF136_0107 was estimated to originate around 2010-2011. CONCLUSION: A novel HIV-1 CRF01_AE/CRF07_BC second-generation CRF called CRF136_0107 was identified among MSM in Heilongjiang, a northeast province of China.

Young MSM changed temporal HIV-1 epidemic pattern in Heilongjiang Province, China.

Background: Human immunodeficiency virus type 1 (HIV-1) epidemic in China is featured by geographical diversity of epidemic patterns. Understanding the characteristics of regional HIV-1 epidemic allows carrying out targeted prevention and controlling measures. This seven-year cross-sectional study was conducted in Heilongjiang, one province of Northeast China, where newly diagnosed infection is fast increasing yearly, but temporal HIV-1 epidemic trend is largely unknown. Methods: Information of 1,006 newly diagnosed HIV-1-infected participants were collected before antiretroviral therapy during 2010-2016 in Heilongjiang province. HIV-1 genotype was identified based on the viral gag and env gene sequences. Recent infection was determined by Limiting-Antigen Avidity assays. Comparison analyses on the median ages, CD4 counts, proportions of stratified age groups and CD4 count groups, and rates of recent HIV-1 infection among different population and sampling times were performed to understand temporal HIV-1 epidemic features. Results: Homosexual contact among men who have sex with men (MSM) was the main transmission route and CRF01_AE was the most dominant HIV-1 genotype. During 2010-2016, the HIV-1 epidemic showed three new changes: the median age continued to decline, the cases with a CD4 count more than 500 cells/µl (CD4hi cases) disproportionally expanded, and the recent HIV-1 infection rate steadily increased. MSM cases determined the temporal trend of HIV-1 epidemic here. Increase of young MSM cases (aged <30 years) made the main contribution to the younger age trend of MSM cases. These young MSM exhibited a higher median CD4 count, a higher proportion of CD4hi cases, and a higher rate of recent HIV-1 infection than cases aged 30 years and more. MSM infected by CRF01_AE virus mostly affected HIV-1 epidemic patterns among MSM population. Conclusion: Young MSM have become a new hotspot and vulnerable group for HIV-1 transmission in Heilongjiang Province, Northeast China. The rapid increase in the number of young MSM cases, mainly those with CRF01_AE infection, changed temporal HIV-1 epidemic pattern here. Measures for prevention and control of HIV-1 infection among this population are urgently needed in the future.

Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence.

Ideal immunogenicity in antigens is a prerequisite to eliciting a sufficiently strong immune and memory response via either DNA or protein vaccines. To improve immunogenicity, efforts have focused on high-level expression of target proteins and on maintaining their natural conformations. In the present work, two trimer motifs (MTQ and MTI) were designed and introduced into a plasmid vector with the tissue plasminogen activator signal peptide (tPA-SP). Next, we examined the efficacy and the efficiency of the two motifs as well as the introduction of tPA-SP and its mutant forms, 22P/A and 22P/G, in facilitating the secretory expression of trimeric proteins in mammalian cells. We found that both trimer motifs could produce the target protein in a trimeric form at a high level. Introduction of tPA-SP 22P/A markedly increased the secretory expression level. The combination of the trimer motif, MTQ, and the signal peptide, 22P/A, may serve as a universal mammalian vector for producing trimeric proteins in vaccine development.

A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin.

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

DNA adjuvant Amiloride conjunct long immunization interval promote higher antibody responses to HIV-1 gp41 and gp140 immunogens.

Recent studies have revealed that the interface of gp120 and gp41 and some parts of gp41 are also critical epitopes for elicitation of broadly neutralizing antibodies. Therefore, potential trimeric gp41 or gp140 immunogen candidates are needed. Previously, we developed a trimer motif MTQ and demonstrated that it could help formation of trimeric gp120 and gp140 proteins. In the present study, we immunized Balb/c mice using trimeric gp41-expressing plasmid for prime and monomeric gp41 or trimeric gp140 protein as well as a mutant (Q577A) for boost. The antibody responses in the context of regimens with various immunization intervals and DNA adjuvants including praziquantel (PZQ), cimetidine (CIM), and amiloride (AML) were evaluated. We found that these three adjuvants were not enough to elicit remarkable specific Abs after gp41 DNA immunization, while AML could significantly promote humoral immune responses after protein boosts. Long immunization interval could induce the specific binding Abs earlier and higher and maintain a high level of Abs in the following 27 weeks after final protein boost. Moreover, two times of protein boosts with DNA adjuvant and a longer time interval achieved a higher titer of specific Abs than three times of protein boosts with a shorter time interval. Q577A mutant was benefit for trimeric gp140 boost in the production of binding Abs but harmful to inducing neutralizing Abs, while this mutant in monomeric gp41 presented the opposite trend which may be associated with the immunogen structures. This study highlights the significance of DNA adjuvant Amiloride and long immunization interval in promoting antibody responses and provides new insights into effective HIV immunization regimen design in the future.

Critical amino acid residues and potential N-linked glycosylation sites contribute to circulating recombinant form 01_AE pathogenesis in Northeast China.

OBJECTIVE: The current study aimed to understand epidemiological feature and critical factors associated with pathogenesis of circulating recombinant form (CRF) 01_AE strains in Northeast China. DESIGN: Compared analysis was made between CRF01_AE and non-CRF01_AE samples to understand the pathogenicity features of CRF01_AE. Further analyses between CRF01_AE samples with high or low CD4 cell counts and between samples with different coreceptor usages were done to explore the possible factors correlating to the pathogenesis of CRF01_AE viruses. METHODS: The genotypes of newly identified strains were determined by phylogenetic analyses using Mega 6.06. Coreceptor usage was predicted by Geno2Pheno algorithm. Potential N-linked glycosylation site (PNGS) number was calculated using the online N-glycosite software. The properties of amino acid sequences were analyzed by the online ProtParam tool. RESULTS: CRF01_AE become the main HIV-1 genotype since 2010. Compared with non-CRF01_AE group, the CRF01_AE group showed a higher proportion of samples with CD4 cell count less than 200 cells/µl. Shorter amino acid length, fewer PNGSs and the presence of a basic motif R/KNXT or NR/KT in V4 correlated to a lower CD4 cell count, and existence or coexistence of Thr12, Arg13, Val21 and Lys33, presence of more than 4 of net charges and lack of the PNGS within V3 favored to the X4/R5X4 coreceptor usage of CRF01_AE viruses. CONCLUSION: CRF01_AE has dominated HIV-1 genotype in Northeast China. Infection with CRF01_AE exhibited a fast disease progression, which may be associated with specific amino acid residues and PNGSs in V3 and V4 regions as well as amino acid length of V4 region.

Identification of a New HIV-1 Circulating Recombinant Form CRF65_cpx Strain in Jilin, China.

This study reported a new HIV-1 circulating recombinant form CRF65_cpx virus isolated from a man who have sex with men (MSM) in Jilin, China. The near full-length genome of this virus was composed of 14 mosaic gene fragments derived from CRF01_AE, subtype B' (Thai B) and subtype C, highly similar to the CRF65_cpx viruses recently identified in Yunnan and Anhui of China. Phylogenetic tree analysis suggested that this CRF65_cpx strain was not generated among MSM in Jilin, but originated in southern regions of China and spread to Jilin by MSM population. The emergence of CRF65_cpx in Jilin indicated HIV-1 epidemic in this area was more and more complicated and the MSM population has become the important source for generation of new recombinant viruses. Real-time surveillance of new HIV-1 infections among MSM population is quite required.

Divergent Primary Immune Responses Induced by Human Immunodeficiency Virus-1 gp120 and Hepatitis B Surface Antigen Determine Antibody Recall Responses.

The development of a vaccine based on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) that elicits potent protective antibodies against infection has been challenging. Recently, we compared the antibody production patterns of HIV-1 Env gp120 and hepatitis B virus surface antigen (HBsAg) to provide insights into how we may improve the protective efficacy of Env-based immunogens. Our previous study showed that HIV Env and HBsAg display different mechanisms of antibody elicitation and that T cells facilitate the responses to repeated immunizations. Here, to elucidate the detailed roles of primary immunization in immune memory response formation and antibody production, we immunized C57BL/6 mice with each antigen and evaluated the development of T follicular helper (Tfh) cells, germinal centers, and the memory responses involved in prime and boost immunizations. We found that after prime immunization, compared with HBsAg, gp120 induced higher frequencies of Tfh cells and programmed death (PD)-1+ T cells, greater major histocompatibility complex II expression on B cells, comparable activated B cells, but weaker germinal center (GC) reactions and memory B cell responses in the draining lymph nodes, accompanied by slower antibody recall responses and poor immune memory responses. The above results suggested that more PD-1+ T cells arising in primary immunization may serve as major contributors to the slow antibody recall response elicited by HIV-1 Env.

Identification of a Novel HIV-1 Unique Recombinant Form Comprising CRF01_AE, Subtype B', and CRF65_cpx Among Men Who Have Sex with Men in Jilin, China.

The current HIV-1 epidemic in China is featured by diverse subtypes and continual emergence of new recombinant viruses. This study identified a novel unique recombinant form (URF), JL16013, among men who have sex with men (MSM) in Jilin, China. The JL16013 virus was different from all known subtypes and set up a distinct branch on the phylogenetic tree. This virus had a CRF01_AE backbone with two subtype B' fragments and one CRF65_cpx fragment inserted into gag, pol, env, and nef regions, suggesting that this novel URF might have originated from the CRF01_AE, subtype B', and CRF65_cpx viruses that were cocirculating in Jilin province. This was the first report of the CRF01_AE/B'/CRF65_cpx recombinant in China. Identification of this URF indicated the severity and complexity of the HIV-1 epidemic among MSM in Jilin province. Timely surveillance of new HIV-1 infections and new recombinants among the MSM population is urgently required.

Live attenuated Salmonella displaying HIV-1 10E8 epitope on fimbriae: systemic and mucosal immune responses in BALB/c mice by mucosal administration.

The HIV-1 membrane proximal external region (MPER) that is targeted by several broadly neutralizing antibodies (BNAbs) has been considered a potential immunogen for vaccine development. However, to date the immunogenicity of these BNAb epitopes has not been made sufficiently adequate. In the present work, we used live attenuated Salmonella as a platform to present the HIV-1 MPER 10E8 epitope in the fimbriae. The insertion of the 10E8 epitope into the fimbriae had no significant influence on the expression and the absorption capacity of bacterial fimbriae, nor on the virulence and invasiveness of the attenuated Salmonella. After oral administration of the vaccine construct to mice followed by 10E8 epitope peptide boost, specific antibody responses in serum and mucosa as well as memory lymphocytes in spleen and plasma cells in bone marrow were induced. We also found that the live attenuated Salmonella vector directed the immunity toward Th1 bias, induced Th1 and Th2 cytokine responses and stimulated significant B cell differentiation into GC B, memory B and plasma cells. Therefore, we propose that the live attenuated Salmonella constitutively expressing HIV-1 BNAb epitopes on the fimbriae will be an effective approach to improving immune microenvironment and enhancing the immunogenicity of HIV-1 epitope vaccines.

Comparison of the patterns of antibody recall responses to HIV-1 gp120 and hepatitis B surface antigen in immunized mice.

To date, we still lack an ideal strategy for designing envelope glycoprotein (Env) vaccines to elicit potent protective antibodies against HIV-1 infection. Since the human hepatitis B virus surface antigen (HBsAg) is representative of effective vaccines that can induce ideal humoral immune responses, knowledge of how it elicits antibody responses and T helper cells would be an useful reference for HIV vaccine development. We compared the characteristics of the HIV-1 Env gp120 trimer and HBsAg in antibody elicitation and induction of T follicular helper (Tfh) and memory B cells in immunized Balb/c mice. Using the strategy of protein prime-protein boost, we found that HIV-1 gp120 induced slower recall antibody responses but redundant non-specific IgG responses at early time after boosting compared to HBsAg. The higher frequency of PD-1hiCD4+ T cells and Tfh cells that appeared at the early time point after gp120 boosting is likely to limit the development of memory B cells, memory T cells, and specific antibody recall responses. These findings regarding the different features of HIV envelope and HBsAg in T helper cell responses may provide a direction to improve HIV envelope immunogenicity.

Molecular Genotyping of HIV-1 Strains from Newly Infected Men Who Have Sex with Men in Harbin, China.

In this study, blood samples from newly HIV-1 infected men who have sex with men (MSM) were collected, and HIV-1 genotypes were identified based on gag p17-p24 and nef gene regions. We found that participants aged from 20 to 40 years old were the major infection group in Harbin. CRF01_AE was the predominant genotype, contributing to 84.7% of HIV-1 infections, followed by subtype B (4.7%), CRF07_BC (3.5%), and subtype B' (Thai B, 1.2%). Moreover, five unique recombinant forms (5.9%) were also identified, including genotypes 01B, 01C, and 01/02. The recombinant CRF01_AE/CRF02_AG was first reported in China. These results suggested that current HIV-1 genotype epidemic among MSM in Harbin is more complicated and that intersubtype recombinants have emerged. Therefore, timely regional epidemiological surveillance of HIV-1 genotype and development of prevention measures for new HIV-1 infections among MSM are quite important.

An HIV-1 envelope immunogen with W427S mutation in CD4 binding site induced more T follicular helper memory cells and reduced non-specific antibody responses.

The CD4 binding site (CD4BS) of the HIV-1 envelope glycoprotein (Env) contains epitopes for broadly neutralizing antibody (nAb) and is the target for the vaccine development. However, the CD4BS core including residues 425-430 overlaps the B cell superantigen site and may be related to B cell exhaustion in HIV-1 infection. Furthermore, production of nAb and high-affinity plasma cells needs germinal center reaction and the help of T follicular helper (Tfh) cells. We believe that strengthening the ability of Env CD4BS in inducing Tfh response and decreasing the effects of the superantigen are the strategies for eliciting nAb and development of HIV-1 vaccine. We constructed a gp120 mutant W427S of an HIV-1 primary R5 strain and examined its ability in the elicitation of Ab and the production of Tfh by immunization of BALB/c mice. We found that the trimeric wild-type gp120 can induce more non-specific antibody-secreting plasma cells, higher serum IgG secretion, and more Tfh cells by splenocyte. The modified W427S gp120 elicits higher levels of specific binding antibodies as well as nAbs though it produces less Tfh cells. Furthermore, higher Tfh cell frequency does not correlate to the specific binding Abs or nAbs indicating that the wild-type gp120 induced some non-specific Tfh that did not contribute to the production of specific Abs. This gp120 mutant led to more memory Tfh production, especially, the effector memory Tfh cells. Taken together, W427S gp120 could induce higher level of specific binding and neutralizing Ab production that may be associated with the reduction of non-specific Tfh but strengthening of the memory Tfh.

Critical amino acids within the human immunodeficiency virus type 1 envelope glycoprotein V4 N- and C-terminals contribute to virus entry.

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.