Neutralization Characteristics HIV-1 CRF01_AE Env Clones from Infections in China

Owing to the increasing prevalence of HIV-1 CRF_01AE, it is necessary to understand the neutralization properties of CRF_01AE and to develop broadly neutralizing monoclonal antibodies (bnmAbs) that can neutralize this virus. The full-length Env gene was cloned from HIV-1 CRF01_AE-infected plasma specimens collected in China and used to establish pseudoviruses. Neutralization phenotypes of the pseudoviruses were characterized with bnmAbs. The neutralizing activities of 11 bnmAbs VRC01, VRC03, IgG1b12 and 3BNC117 (targeting the CD4 binding site); PG9 (targeting the V1V2 region); 2G12 (V3 glycans), PGT135 and 10-1074 (targeting the activity, neutralizing more than 75% of the pseudoviruses; followed by PG9 and 4E10, showing moderate neutralizing activity with neutralization of 50%–60% of the pseudoviruses; whereas the efficacies of the remaining bnmAbs were poor, neutralizing less than 15% of pseudoviruses tested. Env variants of CRF_01AE from one infection also showed significant differences in resistance to neutralization. These characterized HIV-1 CRF_01AE pseudoviruses could be used for neutralization studies and evaluation of vaccines or anti-HIV-1 products in China.


Introduction
Neutralizing antibodies (nAbs) play a critical protective role in viral diseases such as hepatitis, influenza and the novel coronavirus disease 2019 [1][2][3]. At a time when there is no effective vaccine to protect against human immunodeficiency virus (HIV)-1 infection [4], the protective role of nAbs has been reported [5]. These antibodies can be used for the evaluation of vaccine candidates and analysis of HIV-1 epitopes. A three-tier method for the evaluation of vaccine-induced nAbs using pseudoviruses was previously proposed [6], which is safer and more suitable compared with the use of traditional live viruses. This approach also allows for targeted mutation of amino acids and can be used for high-throughput immunogen screening [7].
To analyze each nAb subtype systematically, researchers believe that characteristic pseudovirus panels should be developed for each HIV-1 group M variant [7]. The CRF01_AE variant is mainly sexually transmitted and has gradually become the most prevalent subtype since its discovery in China in the 1990s [8]. As of 2020, CRF01_AE variant infections account for 37.6% of total HIV-1 infections [9], and the number of infections is also increasing in South and Southeast Asia. To evaluate a vaccine against the HIV-1 CRF01_AE variant, the pseudovirus library established by the National Institutes for Food and Drug Control includes some CRF01_AE pseudoviruses [10], and the pseudoviruses constructed in this study can also be included to enrich the previous pseudovirus library.

Cloning Env DNA cassettes and pseudovirus construction
Env DNA fragments were successfully amplified from 13 HIV-positive samples.
The lengths of all amplification products were as expected. The positive clones were identified and co-transfected with the backbone plasmid pSG3 ∆env into 293T cells.
Clones that could form pseudoviruses with appropriate titers were selected from each sample for further analysis. Finally, 36 pseudoviruses were obtained with titers ranging from 4000 to 150,000 median tissue culture infectious dose per mL.
Phylogenetic tree analysis of full-length Env showed that all 36 pseudoviruses belonged to the CRF01_AE subtype (Fig. 1). strains. The newly constructed Env clones are indicated by solid circles, with circles of the same color representing the same plasma source and the rest being reference strain Env clones. Lengths of horizontal branches are drawn to scale (scale bar indicates 0.02 nucleotide substitutions per site), while vertical separations are for clarity only. Values at nodes indicate the percentage of bootstraps in which the cluster to the right was found.

Differences in neutralization of AE variants among bnmAbs
According to the literature [11], the 11 bnmAbs investigated were classified into five categories based on recognition sites, targeting CD4 binding sites (CD4bs), the membrane proximal extracellular region (MPER), the apical V1V2 region, the high mannose glycan on gp120, and the glycan-V3 region. The tested pseudoviruses were found to be more susceptible to bnmAbs targeting CD4bs (IgG1b12, 3BNC117, VRC01 and VRC03) and those targeting MPER (2F5, 4E10 and 10E8) compared with antibodies targeting other sites. The bnmAb PG9, which recognizes the apical region of the Env trimer, showed moderate neutralizing activity for the tested pseudoviruses, while a few pseudoviruses could be neutralized by the 2G12 bnmAb, recognizing high mannose glycans on gp120. bnmAbs recognizing the glycan-V3 region (PGT135 and 10-1074) were the least effective at neutralizing the pseudoviruses used in this study. Of the tested bnmAbs, VRC01 demonstrated the strongest efficacy, neutralizing 100% of the pseudoviruses tested, followed by 10E8 and 3BNC117, which neutralized 78% and 75% of the pseudoviruses, respectively. Both 4E10 and PG9 showed moderate neutralization rates, neutralizing 58% and 53% of the pseudoviruses, respectively; the remaining bnmAbs had poor neutralization rates for pseudoviruses at less than 15%, and PGT135 failed to neutralize the tested pseudoviruses (Table S3). A heat map of the neutralization effect of the bnmAbs on different CRF01_AE-infected patient sera ( Fig. 2A) was constructed to compare bnmAb neutralization efficiencies, and the overall neutralization abilities of the bnmAbs for all pseudoviruses in this study were represented by the geometric mean IC50, with smaller IC50 indicating stronger neutralization ability (Fig. 2B). The results showed that VRC01, 10E8 and 3BNC117 demonstrated most effective neutralization of CRF01_AE variants circulating in recent years, compared with other bnmAbs.

Effect of Env variants on neutralizing activity
Seventeen Env clones were amplified from 2019GX001, and the resulting pseudoviruses had different neutralization characteristics when exposed to bnmAbs, showing escape from single antibodies (

DISCUSSION
The characterization of 36 CRF01_AE pseudovirus variants with 11 bnmAbs identified a number of sites that may affect the neutralization activity of bnmAbs.
Antibody 3BNC117 recognizes a discontinuous epitope on gp120, and its neutralization activity is affected by changes at several sites (including P124, T198, V275, A281, R308, S365, Q428, G459, N462, and G471), of which amino acid changes at the G459 site makes the virus more susceptible to neutralization by 3BNC117 [12]. Antibody 2G12 is a glycosylation-dependent bnmAb that recognizes glycosylated conformational epitopes, including N295, N332, N386, N392, N397 and N448 [13]; these epitopes have been reported to play an important role in the binding of 2G12 to gp120 [14]. Only two of the 36 pseudoviruses used in this study could be neutralized by 2G12, and the 34 pseudoviruses showing no susceptibility to 2G12 had varying mutations at the above-mentioned loci, suggesting that N295 and N332 play an important role in binding. The two pseudoviruses SZ18S0004.3 and 2019GX030.8 that were highly susceptible to 2G12 carried 339N in addition to 295N and 332N, suggesting that the N339 position may also be important for 2G12 recognition of the virus.
The core sequence of the epitope recognized by the 4E10 bnmAb is located at positions 671-676 with the sequence NWFNIT, with additional important binding related sites. Previous reports have shown that three sites-672W, 673F and 680W-are critical for the neutralization activity of antibodies [15], but the relationship between amino acid mutations in the above sites and neutralization quasi-species of escape mutations against bnmAb. For this reason, among others, researchers have tried to use a "cocktail" approach to increase the broad-spectrum and neutralizing activity in bnmAb treatment, which is already available in "3BNC117+10-1074" therapy [17]. According to the results of this study, it was hypothesized that bnmAbs recognizing CD4bs and MPER would have the best neutralizing activity against CRF01_AE pseudoviruses. Among the observed escapes of virus from bnmAbs, the 2019GX001 and 2019GX037 strains both could be neutralized by VRC01, suggesting that the site recognized by VRC01 may be more conserved, and it may be the most suitable bnmAb for the treatment of CRF01_AE.
Meanwhile, 10E8 showed the second strongest neutralizing activity, suggesting that "VRC01+10E8" therapy may be promising for the treatment of CRF01_AE-infected patients in China.
Pseudoviruses, as a novel tool with the advantages of safety, high throughput, and the possibility of targeted amino acid mutations, are gradually being used in vaccine evaluation, and their sensitivity may exceed that of viral infection experiments [18]. We found that HIV-1 CRF01_AE pseudoviruses showed higher susceptibility to antibodies against CD4 binding sites and MPER than antibodies against V1V2 and V3 regions. Among them, three bnmAbs-VRC01, 10E8 and 3BNC117-showed the highest neutralizing potency against CRF01_AE variants.
Considering the high mutagenicity of the HIV-1 virus, combination bnmAb therapy holds promise for successful treatment of HIV-1 infection.

Plasma samples
The Chinese Center for Disease Control and Prevention collected plasma samples from 13 HIV-1 CRF01_AE subtype-infected patients from 2018 to 2019, successfully cloned the full length Env genes from the isolated viruses and used them to construct 36 pseudoviruses (Table S1). All samples were subtyped according to the amplified Env, and the pseudovirus samples were named according to the collection region and serial number.

Amplification, cloning and phylogenetic analysis of HIV Env
Viral RNA was extracted from 140 μL of plasma using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). cDNA was reverse transcribed from total viral RNA using the PrimeScriptTM III first strand cDNA synthesis kit (Takara,kyoto, Japan). Primers were designed (Table S2), and the viral Env DNA fragments were amplified by nested polymerase chain reaction (PCR) using the following conditions. bootstraps in Mega 6.0 was used to construct a neighbor-joining tree, and site-specific analysis was performed using Bioedit.

Pseudovirus preparation, titration, and neutralization assay
Plasmids were transfected into HEK293T cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA), and pseudoviruses were harvested and titrated after 2 days. Neutralizing ability was measured by the decrease in Luc reporter gene expression after a single round of virus infection of TZM-BL cells. These assays were performed as described previously [35].

Statistical analysis
A phylogenetic tree was mapped using Mega 6.0 to understand the phylogeny of the new AE strains, and S-curve testing was performed using GraphPad Prism 8.0 to assess the neutralization titer between HIV-1-positive plasma samples and mAbs.
The shades of color in the map represent the strength of the response, with darker and lighter colors representing stronger and weaker responses, respectively. The ID50 values were converted to base 10 logs before plotting.

Statement
All experimental protocols were approved by National Center for AIDS/STD Control and Prevention, China CDC.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study. All subjects are over 18 years old. Figure 1 Phylogenetic analysis of the newly sequenced HIV-1 Env genes alongside reference strains. The newly constructed Env clones are indicated by solid circles, with circles of the same color representing the same plasma source and the rest being reference strain Env clones. Lengths of horizontal branches are drawn to scale (scale bar indicates 0.02 nucleotide substitutions per site), while vertical separations are for clarity only. Values at nodes indicate the percentage of bootstraps in which the cluster to the right was found. Geometric means of bnmAb neutralization against 36 pseudoviruses were used for comparison; for IC50>10 μg/mL, the graph was calculated with IC50 of 10 μg/mL.

Supplementary Files
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