Systematical analysis of HIV-1 positive sera has revealed that about 5%–25% of HIV-1-infected individuals develop broadly neutralizing antibody (bNAb) response.1–10 In addition, increased neutralization breadth and titers to heterologous viruses have also been noted in long-term nonprogressors.11–13 Apparently, the presence of bNAbs is likely associated with controlling of HIV viremia and disease progression, although less neutralization breadth and titers in long-term nonprogressors have been also found by several studies.14,15
However, it is well recognized that vaccination using HIV-1 envelope cannot successfully induce such antibodies. The knowledge concerning the natures of the envelope glycoproteins related to eliciting bNAbs and the antibody specificities in bNAbs+ individuals are needed urgently. Several studies have been conducted to analyze the antibody specificities in HIV-1-positive plasma displaying neutralization breadth and potency.3–5,16–18 Some reports revealed that neutralization breadth of plasma is achieved by multiclonal antibodies and is rarely mapped to a single epitope.7,19 However, it has also been reported that the specificities of some protective sera were mapped to a limited number of epitopes.20,21 Plasma containing antibodies targeting CD4-binding site (CD4bs), coreceptor binding site, and the membrane-proximal external region of envelope have been identified.2,6,17 To date, epitope specificities of the bNAbs+ sera have still not been elucidated completely.
It has been found that the production of bNAbs may be correlated with viral load and the diversity of HIV-1 envelope proteins.22,23 Recently, a subsets of viruses isolated in the acute stage of HIV-1 infection have been identified as “founder/transmitter.”24–28 Some founder/transmitter envelope glycoproteins are more sensitive to bNAbs,27,28 and the unmutated common ancestor of a mature antibody lineage avidly bound the transmitted/founder Env.24 However, the characteristic of HIV-1 Envs correlating to their capacity of eliciting bNAbs is still unknown.
The profile of antibody responses in Chinese HIV-1-infected individuals has been rarely studied. Very recently, a study reported that 15% of HIV-1-infected patients displayed cross neutralization in a Beijing homosexual male cohort infected with subtypes B, BC, or AE recombinant strains.29 Bao et al8 found that 18 of 117 HIV-1 sera (CRF07_BC) possess cross-neutralizing activity against 3 viruses and that the sera from more asymptomatic individuals showed better neutralization compared with the sera from symptomatic individuals. However, lacking in comprehensive information about the profile of antibody responses and the characteristics of the envelope proteins from Chinese bNAbs+ individuals restricts the elucidation of the natures of bNAb production.
Present work focused on the elucidation of the profile of NAb responses in Chinese HIV-1-infected individuals from Xinjiang and Guangxi provinces, where CRF07_BC and CRF01_AE are the representative subtypes. These 2 subtypes are dominant in China accounting for 66% of HIV-1 infection in China.30 We also focused on the specificities of viral envelope genes and their encoded amino acid sequences of broad neutralizers. The results would provide valuable data for designing effective vaccines to prevent the epidemic of the unique circulating recombinant HIV-1 subtypes in China.
Plasmas were collected from HIV-1-infected individuals in China. Eligible participants were those who infected HIV-1 for at least 3 years and received no antiretroviral therapy before the sampling date. Six hundred eleven samples were from Xinjiang province and 249 samples from Guangxi province. Plasmas were stored at −80°C and inactivated at 56°C for 1 hour before assays.
The study was approved by the Harbin Medical University Ethics Committee (HMUIRB20140002). No informed consent from participants was obtained as the data were analyzed anonymously.
Envelope-pseudotyped viruses (see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A567) were produced by cotransfection of an env-expression-plasmid and a plasmid pSG3ΔEnv into 293T cells. The viral infectivity was titrated in TZM-bl cells as described.31 Thirty-three pseudoviruses of 5 HIV-1 subtypes including subtype B, C, CRF07_BC, B′, and CRF01_AE were used. The international reference viruses of subtype B and subtype C have been reported.31,32 The viruses including subtypes CRF01_AE, CRF07_BC, and B′ were obtained from infected individuals in various areas of China.
BnAbs 2G12,33 b12,34 PG9, PG16,17 2F5,35 VRC01, VRC03,21 447-52D,36 HJ16,37 and 4E1038 were provided by NIH AIDS Research and Reference Reagent Program.
Neutralization assay was carried out using a single-round pseudovirus infection in TZM-bl cells.31,39 First, we detected the neutralization activities of the plasmas (expressed as the percentage of neutralization, NP) at dilution of 1:100 against 11 Env-pseudoviruses from 3 subtypes (subtypes B, C, and CRF01_AE). Subsequently, the neutralization profile of the plasmas identified as broad neutralizers was examined by detecting the 50% inhibitory dose (ID50) against all the 33 pseudoviruses. And the neutralization sensitivities of Env clones were examined by detecting the 50% inhibitory dose (IC50) of bnAbs against these clones.
Single-Genome Amplification of Envelope Genes
Viral RNAs were extracted from plasma samples using QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) and subjected to first-strand complementary DNA (cDNA) synthesis using Primescript II First Strand cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). Single-genome amplification was performed to avoid artificial recombination and resampling of viral genomes as described in earlier publications.26,27,40 Briefly, 1 to 3 serial dilution of cDNA was made, and nested polymerase chain reaction amplification was performed (10 reactions per dilution) using the PrimeSTAR HS DNA Polymerase (Takara Biotechnology) to determine a dilution at which 30% or less of positive amplifications were obtained. Then, the env gene cassettes were amplified at this dilution of cDNA. All the primers were listed in Table S2 (see Supplemental Digital Content,http://links.lww.com/QAI/A567).
Sequence Alignments and Signature Analysis
Multiple env genes from each HIV-1 elite neutralizers were obtained. We selected all the HIV-1 CRF07_BC env sequences from HIV Sequence Database (http://www.hiv.lanl.gov) (n = 75), which were considered as nonelite neutralizers because only 1% of HIV-1-infected individuals produce neutralizing antibodies across at least 4 subtypes with IC50 ≥300 and defined as elite neutralizers.5 The Env amino acid sequences were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clusta-lw2/). The number of potential N-linked glycosylation sites of the deduced amino acid sequences was calculated using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). The net charge was analyzed using GeneRunner version 3.05. The differences of the amino acid lengths, potential N-linked glycosylation sites, and net charges in the sequences between the elite neutralizers and the nonelite neutralizers were assessed using the Wilcoxon rank-sum test.
To determine the Env amino acid sequence markers associated with broad neutralization, we compared the sequences from the 2 groups. We scanned each amino acid of the aligned envelope sequences of the 2 groups except the subregions, which were unable to be aligned exactly containing more than 10% gaps (for instance, tip of V4).41 The Fisher exact test was used to identify statistically interesting sites. All P values were 2-sided, and a P value <0.05 was considered statistically significant.
Nucleotide Sequences Accession Numbers
GenBank accession numbers of the env gene sequences are KC807912- KC807968, as shown in Table S3 (see Supplemental Digital Content,http://links.lww.com/QAI/A567).
The Neutralizing Profile of Plasmas Against Pseudoviruses of Subtypes B, C, and CRF01_AE
We first examined the antibody responses of the plasmas against a panel of pseudoviruses including 4 of subtype B, 4 of subtype C, and 3 of CRF01_AE (see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A567) to identify the neutralization profile of the plasmas. Intrasubtype neutralization against subtype B or C was defined as neutralization of at least 2 of the 4 viruses from each subtype. Intersubtype cross neutralization against subtypes B and C was defined as neutralization of at least 2 viruses from both subtypes. Broadly neutralizing plasmas were defined as intersubtype neutralizing plasmas and could neutralize at least 1 CRF01_AE virus.29 The percent inhibition (NP) at plasma dilution of 1:100 was reported to indicate the relative strength of neutralizing activity while inhibition less than 50% was considered negative.42
In Xinjiang, 54.0% and 35.7% of the plasmas displayed intrasubtype neutralization against subtype C and subtype B viruses, respectively (Table 1). Of the plasmas from Guangxi, much lower proportions of plasmas displayed intrasubtype neutralization against subtypes B (11.2%) and C (11.6%).
We also found that 29.1% of the plasmas from Xinjiang and 5.2% of the plasmas from Guangxi displayed intersubtype neutralization to subtype B and C viruses. From Xinjiang, 10.6% of the plasmas displayed broad neutralization activity against the 3 subtypes, whereas only 2.4% of the plasmas from Guangxi displayed broad neutralization. However, there was very limited proportion (0.8%) of plasmas displaying cross neutralization with high potency (NP >80%) against 3 subtypes in Xinjiang, whereas none of the plasmas from Guangxi had such activity at dilution of 1:100. In general, the intersubtype- and cross-neutralization rates were 22.2% and 8.3%, respectively (Table 1).
Of the 611 Xinjiang plasmas, 12% and 8% did not show neutralization activity (NP <50%) to any of the 4 viruses from subtype B nor C viruses at dilution of 1:100. The rates were 15% and 14%, respectively, for the plasmas from Guangxi.
The Breadth and Potency of Neutralization of the Plasmas Developing Elite Activity
Among the plasmas displaying intersubtype neutralization against 3 subtype viruses, we were able to examine the neutralizing activity of 23 plasmas with sufficient quantity for the neutralization assay against a panel of 34 pseudoviruses including 33 HIV-1 pseudoviruses and 1 control, VSV-G pseudovirus (Fig. 1; see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A567). We identified 14 plasmas showing high potency of cross neutralization against the viruses of 5 subtypes, thus defined as bNAb+ plasmas. All 14 HIV-1 bNAb+ plasmas neutralized SF162 (tier 1) with extra-high potency (geometric mean ID50 titer = 1:4179), except plasma 09467 (Fig. 1). The geometric mean titers (GMT) of the neutralization of all the bNAb+ plasmas against viruses of 5 subtypes ranged from 1:203 to 1:388. Among them, 6 plasmas showed higher neutralizing activities (GMTs were higher than 1:300).
Meanwhile, all the bNAb+ plasmas neutralized the viruses from China, which confirmed the cross neutralization of the plasmas with slightly subtype-restricted neutralization. The rank order of intrasubtype neutralizations (neutralization of SF162 was not included) was CRF01_AE (1:218) < B (1:253) < CRF07_BC (1:310) < C (1:317). The potency of intrasubtype neutralization against subtype B′ was not ranked because of limited numbers of viruses tested. Of note, the plasmas displayed higher rank of neutralization potency against subtype CRF07_BC and subtype C viruses. One plasma, 09330 from Xinjiang, could neutralize viruses of all of the 5 subtypes with high potency, with GMT values >1:350.
In summary, we identified 14 individuals whose plasmas can neutralize 70% of 33 HIV-1 viruses from 5 subtypes with ID50 >1:100. Among them, 5 plasmas (0501, 0651, 0901, 09330, and 09432) displaying breadth neutralizing 90% of 33 viruses and 6 (1081, 09278, 09330, 09432, and GX752, GX757) are potent which with ID50 >1:300 (Fig. 1; see Figure S1, Supplemental Digital Content,http://links.lww.com/QAI/A567). We defined these individuals as elite neutralizers.
Interestingly, these plasmas displayed good cross-neutralization activities against the viruses from South Africa but with lower potency against those from Zambia. The plasmas had good cross-neutralization activity against the subtype B viruses from Italy (2/2) and United States (3/7) with high potency (GMTs >1:300, ranged 1:301–1:490, SF162 not included). And, 1 B′ virus (CHLJBF06044) was highly sensitive to all the plasmas (GMT = 1:847). Only 1/4 of the CRF01_AE viruses were highly sensitive to the plasmas (GMT = 1:582).
Sequence Diversity and the Unique Characteristics in Variable Regions of Envelope Glycoproteins From the Elite Neutralizers
To characterize the virus env quasispecies from the broad neutralizers, we successfully amplified 56 envelope genes from 6 elite neutralizers (GX752, GX757, 0031, 0651, 1081, and 09466) (see Table S3, Supplemental Digital Content,http://links.lww.com/QAI/A567). The envelope gene sequences were classified as subtype CRF07_BC (from 0031, 0651, 1081, and 09466) and C (from GX752 and GX757).
We analyzed the differences in the length, the N-linked glycans, and the net charges of the variable regions between the envelope sequences from elite neutralizers and those from HIV Sequence Database, which were considered from nonelite neutralizers. We found that the differences of the length, the N-linked glycans, and the net charges exist dominantly in V1, V2, or V4 regions (Fig. 2). The envelopes from elite neutralizers had longer V2 and V4 regions and more N-linked glycans in V4 region (P = 0.012), and fewer net positive charges in V4 region (P = 0.047). However, there was no significant difference in the number of N-linked glycans within V1 regions between the 2 groups, although more glycans in V1 have been reported in the elite neutralizers.22
The Possible Signature Epitopes in Viral Envelopes of the Elite Neutralizers
To explore the signature epitopes that may contribute to developing potent bNAb responses, we compared the sequences from the 2 groups. We scanned each amino acid of the aligned envelope sequences of the 2 groups except the subregions, which were unable to be aligned exactly.
First, we focused on the epitopes of well-clarified bNAbs including CD4bs (IgG1b12, VRC01, and VRC03), 2G12, PG9/16, 2F5, 4E10.43 It was found that the envelopes derived from elite neutralizers tended to be more conservative at 4 amino acid residue positions (V455, Y173, N674, and S676) within CD4bs, the epitopes of PG9/16, and 4E10 (Table 2). For instance, 68.4% of the envelopes from elite neutralizers but only 34.7% of those from nonelite neutralizers had V455 within CD4bs epitope (P < 0.01). Similarly, Y674 and S676 were found in 59.6% of the elite neutralizers, whereas they were found in only 24% (P < 0.01) and 12% (P < 0.01) of the nonelite neutralizers. We also found other 11 potential signature epitopes outside the known neutralizing epitopes in C2, C3, and C4 regions, which were considered to be potential unique features of the envelope glycoproteins for the Chinese elite neutralizers. The sequences within the other envelope regions derived from elite neutralizers did not possess unique features.
The Sensitivities of the Env Pseudoviruses From Some Elite Neutralizers to Known Monoclonal bNAbs
We were trying to obtain the full-length env gene quasispecies clones from the elite neutralizers to test the neutralizing sensitivity of these Env quasispecies to the well-characterized monoclonal bNAbs. We were only able to get the functional env gene clones from the neutralizers 0651, 1081, and 0031, respectively, because of the limitation in sample amount or single-genome amplification difficult. We found that Env quacispecies from 0651 were highly sensitive to PG9 and PG16. Some of them were also sensitive to VRC01, HJ16, and 4E10 (Table 3). Moreover, most of the Env quacispecies from 1081 to 0031 also showed the sensitivity to PG16 and 4E10. The Envs from 09277, a nonelite neutralizer, were resistant to all the bNAbs, but except only 09277-7, which showed the sensitivity to PG16. All the Envs were resistant to the bNAbs b12, 2G12, and 2F5.
Understanding the cross-reactivity profile and specificities of serum antibodies from Chinese HIV-1-infected individuals will provide valuable information for the design of vaccines and the strategies to prevent HIV-1 transmission in China. A nationwide investigation focusing on the neutralizing response is needed.
Epidemiologic studies have revealed that China is one of the countries with the most abundant HIV-1 subtypes.30 However, more than 90% of HIV-1 infection involved only 3 subtypes including recombinant BC (CRF07_BC and CRF08_BC), B′ (Thai B), and CRF01_AE, accounting for 50%, 29%, and 16% of all infections in China, respectively.30 CRF07_BC represents the largest percentage of infections in China and is responsible for more than 90% of new HIV infections in Xinjiang Province.44 In Guangxi Province, recombinant CRF01_AE, subtype C, and a variety of recombinant forms of HIV-1 have been found.45,46 However, CRF01_AE is a dominant subtype because 67% of the HIV-1 infections were caused by CRF01_AE in this region.45 While in the central China, the dominant HIV-1 subtype is subtype B′. For instance, 96% of HIV-1 infections in Henan Province are caused by this subtype.47,48 Therefore, knowledge about the profile of antibody responses against the above 3 subtypes reflects the situation in about 95% of HIV-1 infections in China and would be very helpful to design HIV-1 vaccines covering the most infection in China. Present work is the first study of antibody response profile using large scale of specimens in the 2 regions with high HIV-1 infection prevalence where subtypes are representative.
An analysis of 117 HIV-1 serum samples (derived from CRF07_BC infected individuals) revealed that 15.4% of the sera at a dilution of 1:10 neutralized 3 primary virus isolates from subtypes B′, C, and A/G.8 Twenty-nine percent of HIV-1 subtype B′ positive plasma samples neutralized 80% of 25 viruses from subtypes B, C, A, CRF07_BC and CRF01_AE, with ID50 >1:20.9 Zhang et al29 have found that about 15% of HIV-1-infected patients with a variety of subtypes displayed cross-neutralization activities against viruses from 4 subtypes at dilution of 1:20. These few studies using small sample size or based on the potency definition at the serum dilution of 1:10 to 1:20 have unveiled the neutralization characteristics of Chinese infected individuals.
An international collaborative investigation has found that 36% of 97 HIV-1 plasmas from Rwanda and Zambia can neutralize at least 1 pseudovirus from 4 of the 5 subtype/CRF groups with ID50 ≥150.5 A multiple-country collaborative study using samples from Africa, Europe, and Australia has revealed that 34% (463 serum samples) showed broad neutralization with ID50 ≥100 against 4 or more virus groups. Lower proportion of broadly reactive antibodies (20%) with an ID50 ≥100 has been also found.10 We chose subtypes B, C, and AE as the virus panel to clarify the profile of plasma neutralization because the 3 subtypes are the most prevalent subtypes and account for about 95% of HIV-1 infection in China.30 We defined effective neutralization at plasma dilution of 1:100 to hit more strict definition of effective neutralization. To date, the protective serum titer in HIV-1 infection is still unknown but more strict potency definition of neutralization may reflect the higher capacity of plasma neutralization and may provide true protection titer needed.
We found that the proportion of plasmas displaying cross neutralization against B, C, and AE from Xinjiang is as high as 10.6%. This suggests that the chronic infection in the Xinjiang population causes strong and wide cross neutralization. We also identified elite neutralizers in Chinese infected populations. Further extensive analysis of the germline B cell receptor (BCR) from the elite neutralizers identified in this study may provide the evidences for the antibody maturation in the infected individuals in China. In recent years, successful isolation of a series of bNAbs from elite neutralizers and the bNAbs that neutralize 70%–90% of circulating isolates raises new hope in effective control of HIV-1 infection by immunization.5,7,21,49,50 Furthermore, clarification of the Env specificities associated with producing bNAbs in the elite neutralizers will also provide insights for rational design of vaccines against the infection in Chinese populations.
Identifying the viral epitopes associated with the bNAb responses in the bNAbs+ individuals is helpful for designing of efficient HIV vaccines that are able to induce bNAbs. The epitopes targeted by some well-identified bNAbs have been defined but about 50% of the specificities of antibodies in bNAbs+ plasmas have not been elucidated.5,7,21,49,50 It has been found that bNAb+ individuals have longer V1 or/and V2 regions,22,51,52 which is consistent with our findings. Furthermore, we have found that V1 regions have more positive charges. The combined C3 and V4 regions form important structural motifs and the epitopes of which are major targets of the early autologous neutralizing response in HIV-1 subtype C infection.53 The variation within V4/V5 of envelopes causes immune escape from neutralizing antibodies.53 Present and others works54,55 confirmed that the highly variable V4 regions contribute to bNAb responses. The V4 regions of the elite neutralizers have more negative charges, longer length, and more N-linked glycans comparing to that of nonelite neutralizers. However, there is no any single signature within V4 regions has been found, maybe because of extremely high variation in the V4 (data not shown).
We also found 4 signatures from elite neutralizers, which are potentially associated with neutralization sensitivities to PG9/16, CD4bs, and 4E10. It was verified that the Envs of elite neutralizers displayed the sensitivity to the bNAbs PG9, PG16, and 4E10. Most notable among these, 0651, is sensitive to all the bNAbs except b12, 2G12, and 2F5. This implies that not only the plasmas from the elite neutralizers have broadly neutralizing activities but the epitopes of the Envs to the known monoclonal bNAbs are also well exposed. Furthermore, it seems that Envs from HIV-1 CRF07_BC are completely resistant to 2F5 and less than half are sensitive to b12 or 2G12 (Table 3). This study suggested that even the Envs from the elite neutralizers have the similar trends because of the similar mutations at the epitopes targeted by the bNAbs.56,57 We also found that other 11 potential signatures from elite neutralizers are within C2, C3, or C4 regions, outside the known neutralizing epitopes. We could not conclude that they are associated with the broad neutralization phenotype of the elite neutralizers. The significance of these amino acids as the signatures for the broad neutralization activities may be explored using a site mutation strategy.
In this study, we clarified the profile of neutralizing antibody responses in HIV-1-infected individuals from Xinjiang and Guangxi of China, the 2 regions where dominant HIV-1 subtypes are CRF07_BC and CRF01_AE and account for about 66% of HIV-1 infections in China. We found that broad neutralization response against the virus mainly related to envelope variation within V1, V2, and V4. Meanwhile, unique characteristics of the envelope glycoproteins from the Chinese elite neutralizers have been found. This is the largest and most-comprehensive data set of neutralization specificities in HIV-1-infected individuals in China. It will provide insights into vaccine design and prevention strategies in China.
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