Vaccine efficacy in the Thai RV144 vaccine trial was 31%1 and stimulated an intensive worldwide effort to identify the cellular and humoral immune responses associated with this protective effect.2–7 CRF01_AE accounted for 91.7% of RV144 HIV-1 infections,8 but when compared with the large panels of subtype B and C viruses available, the CRF01_AE HIV-1 isolates currently represent only ∼10% of the LANL sequence database.
To assess neutralizing antibody responses, 2 standardized cell-based assays have been developed, the TZM-bl assay,9–11 and, more recently, the A3R5 assay.12,13 Although both assays use cell lines as the target for HIV-1 infection and luminescence as a reporter for target cell infection and both cell lines express the receptors required for HIV-1 infection (CD4, CXCR4, and CCR5), important differences exist between these 2 models. The TZM-bl cell line, derived from epithelial HeLa cells, expresses firefly (FF) luciferase on infection and has been widely used with envelope (Env)-pseudotyped viruses.10,14,15 To facilitate infection, the TZM-bl cell line was engineered to express CD4 and CCR5 at higher physiologic levels16 that are observed for CD4+ T lymphocytes found in vivo.12,17
With the development of HIV-1 full-length replication-competent infectious molecular clones (IMCs) expressing the Renilla reneformis luciferase gene (LucR),18,19 a second cell-based assay using A3R5 lymphoblastoid target cells that naturally express CD4 and CXCR4 and are engineered to express CCR5 in copy numbers similar to that observed on human peripheral blood mononuclear cells (PBMCs) was developed.13,20 However, with previous studies showing differences in neutralization sensitivity between the 2 cell-based assays, uncertainty remains regarding which assay best reflects the events that occur in vivo and might eventually serve to measure antibodies that correlate with protection.
Here, we present the development of 14 Thai CRF01_AE full-length HIV-1 constructs and their neutralization profiles. Thai CRF01_AE envelope genes (envs) were cloned into a novel and highly functional CRF01_AE IMC backbone expressing LucR, resulting in fully functional virions that can productively infect permissive cell lines and natural HIV-1 target cells. The neutralizing activities of monoclonal antibodies (mAbs) and polyclonal sera were measured in both TZM-bl and A3R5 assays. We found that the neutralization susceptibility of these IMCs was greater in A3R5 cells as has been previously shown.18 In addition, comparison of serum antibody-mediated neutralization in the TZM-bl versus A3R5 assays showed little or no correlation.
The TZM-bl cell line10 and the 293T/17 human kidney cell line (CRL-11268) were obtained through the NIH AIDS Research and Reference Program, and from the American Type Culture Collection (ATCC), respectively. Adherent cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (PAA laboratories), 1% L-glutamine, and 1% penicillin/streptomycin (Gibco/BRL). The A3R5.7 cell line20 was made in our laboratory and maintained in RPMI 1640 growth medium supplemented with 15% heat-inactivated fetal bovine serum (PAA laboratories), 1% L-glutamine, and 1% penicillin/streptomycin (Gibco/BRL) and 600 µg/mL (active) geneticin (G418, Gibco/BRL).
With the exception of TH023, CM235, and CM244, isolated from infected PBMC cocultures, CRF01_AE HIV-1 envelopes were retrieved by single genome amplification (SGA) and sequenced from plasma of Thai infected subjects.21 Full viral genomes were also retrieved and cloned to generate full-length IMCs. One particular IMC, 40061, with the highest replicative capacity was selected to serve as CRF01_AE backbone, and the entire gp160 coding sequences were then cloned as previously described.18 Briefly, the cassette for the LucR gene was inserted in the full-length molecular clone, between the env and nef DNA sequences by multiple rounds of fusion polymerase chain reaction and unique enzyme restriction sites, AarI and BglI. The backbone was then further engineered to express the MluI restriction site that is not naturally present. For chimeric constructs, purified env inserts were amplified with primers containing the MluI and BsiWI sequences, digested with the required restriction enzymes, and ligated into the backbone.LucR. Final constructs were sequenced for verification.
Infection and Tropism
Viral stocks were prepared in 293T cells and each stock was titrated in TZM-bl10,11 and A3R520 cell lines to determine TCID50 (50% tissue culture infectious dose). A cut-off value of 3 times over background (uninfected cells) relative light units (RLUs) for FF was implemented, as described elsewhere.14 For LucR, the cut-off was determined as being 3 × [(average of negative wells) + 3SD].18 RLUs were measured on a Victor X light Luminescence counter (Perkin-Elmer) with an exposure time of 0.1 s/well.
Inhibition of HIV-1 infection with different mAbs and sera was analyzed in TZM-bl and A3R5 cells using the 14 chimeric IMCs. TZM-bl and A3R5 neutralization assays were performed as previously described.17,20 Neutralization was measured as the reduction in FF RLU and/or LucR RLU in the presence of serial antibody/serum dilutions. Viral input used in both assay platforms was normalized to a dilution expected to produce a level of RLU at least 10 times above cut-off value.17 The reciprocal titers were derived through averaging values from 2 independent assays for which the IC50 values were within 3-fold range. For our study, soluble CD4 (sCD4) and a total of 11 mAbs recognizing different Env regions were tested: VRC01, 3BNC117, and b12 [CD4 binding site (CD4bs)]; PG9 and PG16 (V1/V2 region); PGT121 and PGT126 (V3 region); 2F5, 4E10, and 10E8 (MPER); and glycan-dependent mAb 2G12. All reagents were obtained through the NIH AIDS Research and Reference Program. Pools of sera from subjects infected with HIV-1 CRF01_AE or subtype B sera were also investigated.17 Serum IgG was depleted using protein G sepharose 4 Fast Flow (GE Healthcare Life Sciences, Marlborough, MA).
DNA sequences were assembled and analyzed using Sequencher version 5.0 (Genecodes Inc., Ann Arbor, MI). Statistical analysis was performed using GraphPad Prism 6.0 software. Wilcoxon matched-paired test was used to assess significant differences of the neutralization sensitivity. Correlation coefficients were evaluated using linear regression.
Newly Developed IMC
We had previously developed IMCs to swap and express HIV-1 env genes as replication-competent viruses in subtype-matched and nonmatched HIV backbones and have shown that non-env genes may impact neutralization profiles.18 At the time these IMCs were engineered, only one CRF01_AE IMC was available, which had been isolated from multiple-passaged PBMCs22 and showed moderate replicative capacity. We have now developed several CRF01_AE full-length IMC, directly derived from plasma of infected Thai subjects using SGA. Among those, the construct showing the highest replicative capacity in TZM-bl and A3R5 cells, 40061, was selected (Supplemental Digital Content Table 1) and engineered to encode (1) LucR and (2) to express gp160 of exogenous HIV-1 env,18 generating a panel of 14 CRF01_AE envs expressed into fully replicative 40061.LucR subtype-matched backbone. Furthermore, 40061.LucR showed productive infection in human PBMCs and monocyte-derived macrophages expanding the scope of immunological assays to HIV-1 natural target cells (Supplemental Digital Content Table 1, http://links.lww.com/QAI/B133).
The CRF01_AE envelope panel chosen was exclusively from Thailand and included mainly Envs derived from recently infected individuals. Among them, 3 were isolated in early Fiebig stage I/II, 2 were isolated in later Fiebig stage I/II, and 3 were from Fiebig stage III-V (Table 1). Those envelopes were SGA derived from plasma of subjects infected between 2005 and 2010. Finally, 3 Envs from subjects isolated early in HIV-1 Thai epidemic, between 1990 and 1992, were added to our panel (Table 1); these envelopes were isolated from PBMC cocultures and have been previously referenced as chronic viruses.23 All virus stocks demonstrated robust viral replication in TZM-bl and A3R5 cells. Most IMCs had higher titers in TZM-bl cells with an average TCID50 of 2 × 106 as compared with 1 × 105 in the A3R5 cell line (Supplemental Digital Content Fig. 1, http://links.lww.com/QAI/B133).
Higher Sensitivity of HIV-1 Neutralization in A3R5-Based Assays
Neutralization sensitivity of the CRF01_AE IMC panel was tested in TZM-bl and in A3R5 cells against sCD4 and an array of mAbs targeting the 4 major antigenic regions on HIV-1 envelope as well as pooled HIV-1 positive sera; individual results are depicted in Table 2. We compared the neutralization results obtained in both assays by pairing all IC50 (mAbs) and ID50 (sera) data and found that neutralization sensitivity to mAbs (Fig. 1A) and to sera (Fig. 1B) was significantly higher in A3R5 than in TZM-bl cells (P < 0.0001). Although a strong correlation of neutralization susceptibility against mAbs between the 2 cell-based assays was found (R2 = 0.7 and P < 0.0001 Fig. 1C), a low to moderate correlation was observed with sera (R2 = 0.17 and P = 0.03 Fig. 1D). To investigate further if the increased sensitivity in the A3R5 assay is mediated by non-IgG factors, we measured neutralization sensitivity of 3 IMCs using untreated and IgG-depleted sera in the A3R5 assay and observed a significant reduction in activity with the IgG-depleted sera (P = 0.03; Supplemental Digital Content Fig. 2, http://links.lww.com/QAI/B133). The low levels of detectable neutralizing activity in some IgG-depleted serum assays could be due to (1) the presence of residual IgG that was not quantified, (2) the presence of potentially neutralizing serum IgA that would not have been fully removed by the protein G sepharose beads, or (3) the presence of potentially neutralizing degraded IgG Fab fragments that would have also not been fully removed by the protein G sepharose beads. However, the neutralizing activity was reduced by 98.8%–99.8% compared with the matched untreated serum, indicating that the majority of the activity was IgG mediated.
We then investigated if mAbs against any particular envelope domain were specifically responsible for the increased neutralization sensitivity observed in A3R5 versus TZM-bl cell-based assay. A panel of mAbs targeting the 4 different antigenic regions on HIV-1 envelope, chosen as described in the Methods and Materials, was significantly more potent in the A3R5 cell-based assay (Fig. 2A; P values range 0.0078 to <0.0001). HIV+ CRF01_AE and subtype B pooled sera were also evaluated (Fig. 2B), with similarly higher neutralization observed using A3R5 when compared with TZM-bl cell targets (P = 0.0005 and P = 0.001, respectively). CRF01_AE viruses were more sensitive to neutralization by matched subtype pooled sera in both cell-based assays, as seen in previous studies.24,25
Higher neutralization susceptibility in A3R5 cells is independent of the virus stage, year, and mode of isolation.
It has previously been shown that viruses isolated in the early years of the HIV epidemic were more sensitive to neutralization than recently isolated viruses.26,27 Previous studies have yielded conflicting results regarding neutralization susceptibility of acute and chronic viruses.24,28–30 As described in Table 1, our panel of CRF01_AE included isolates of different stages of the viral infection and from different times in the Thai HIV epidemic history, ranging from 1990 to 2010. Our panel can be categorized into 3 groups of viruses: early Fiebig stage I/II, late Fiebig stage I/II, Fiebig stage III-V, and one group of chronic viruses (Fiebig stage VI). In this study, we analyzed the combined neutralization data obtained in both cell-based assays against the 11 mAbs and sCD4. Although higher sensitivity was observed in A3R5 cells, a similar trend of neutralization was observed among the 4 groups of viruses in TZM-bl cells (Fig. 2C). Late Fiebig stage I/II viruses were less sensitive to neutralization than chronic (P < 0.0001) and, unexpectedly, less sensitive compared with early Fiebig stage I/II viruses (P = 0.0002 and 0.0003, respectively). Although there was a trend toward higher neutralization sensitivity with HIV-1 from the early Thai epidemic (1990's) in TZM-bl and A3R5 cells, no statistical differences were found except between early Fiebig stage I/II and late Fiebig stage I/II viruses in A3R5 cells and between late Fiebig stage I/II and chronic viruses in TZM-bl cells (Fig. 2D). The increased neutralization sensitivity observed in A3R5 was neither linked to the year of virus transmission nor to the stages of infection as represented in Figure 3, which depicts the neutralization sensitivities of individual viruses against the panel of mAbs. Interestingly, mAb neutralization of 816763 revealed the unique case of higher TZM-bl sensitivity (Fig. 3; P = 0.03).
The need for replication-competent IMC that can be used in a variety of cell types and standardized immune assays has become increasingly important. These molecular constructs express the entire gp160 Env, have full replicative capacity, and preliminary results have shown that they can be used in other immunological assays such as antibody-dependent cell-mediated cytotoxicity31 (Ferrari G, personal communication). Here, we present a panel of novel reporter IMC to assess neutralization susceptibility of CRF01_AE envelopes and to better evaluate the immune responses against CRF01_AE HIV-1 using TZM-bl and A3R5 cell-based assays. Because we have previously shown that non-env genes may affect neutralization sensitivity,18 a CRF01_AE IMC was selected to serve as an HIV backbone for use with CRF01_AE envelopes for assessing vaccine responses in trials conducted in Thailand and countries where CRF01_AE HIV-1 is prevalent. This IMC was SGA derived from a plasma sample of a Thai subject 7 days after the last negative blood test, qualifying it as a transmitted/founder virus. This virus showed a high replicative capacity and was never passaged in the laboratory, making it a suitable backbone vector that should perform at a level similar to naturally transmitted virus to express heterologous CRF01_AE Thai Envs.
IMCs in this study were characterized for sensitivity to neutralization by mAbs and sera in the 2 standardized assays, TZM-bl and A3R5 cell-based assays. Greater neutralization sensitivity was observed in A3R5 compared with TZM-bl cells for all the reagents tested, supporting previous reports.13,32 However, a significant correlation between assays was only observed with monoclonal antibodies (R2 = 0.7, P < 0.0001). These data imply that, despite the higher neutralization sensitivity of the A3R5 assay, both cell lines measure similar patterns or relationships between mAb activities against these IMCs. Similar results were reported in the analysis of the RV144 and Vax003 HIV-1 vaccine efficacy trials, where serum neutralizing activity was detected in the A3R5 assay but not the TZM-bl assay when using the same CRF01_AE IMC (16). Because of the heightened sensitivity of the A3R5 assay for detecting neutralizing antibodies, discrepancies between assay platforms may be more apparent when neutralization-resistant, tier 2 virus stocks are used. In addition, differences in monoclonal antibody neutralization of cell-free versus cell-associated HIV-1 have been reported for the TZM-bl and A3R5 assay.33 In both assays, cell-free virus was found to be more sensitive to neutralization when compared with cell-associated virus; however, this was dependent on the specificity of the mAb tested.
No correlation (R2 = 0.002) was observed when comparing the serum titers obtained using TZM-bl cells versus PBMC, which express CCR5 levels similar to those of A3R5 cells,20 and higher neutralization sensitivity was observed using PBMC compared with TZM-bl cells, using both mAbs34 as well as sera.17 This is not surprising, particularly with respect to polyclonal sera, for which the full content of the antibody repertoire may be largely unknown, and polyclonal sera have varying epitope specificities with potentially differing affinities and/or valencies. Antibody–virus–host cell interactions may also be heavily influenced by target cell adherence, primary and coreceptor densities, and the presence or absence of other host cell molecules at the cell surface and/or incorporated into virions; all these parameters may affect neutralization read-outs.
We confirmed that the more potent A3R5 activity observed in this study was due to IgG-mediated HIV neutralization and not a result of an artifact registered only when using A3R5 cells. The increased neutralization sensitivity observed in the A3R5 assay was not linked to the year of virus transmission or to the stages of infection. Indeed, with the exception of 816763, all viruses were more susceptible to mAb neutralization in A3R5 than in the TZM-bl cell-based assay. Chronic viruses from the years 1990–92 were more sensitive to neutralization than the more current viruses, in both assays. The findings on subtype B HIV-1 showing an increase in neutralization resistance over a period of 20 years have also been reported.26,27 With the limited panel of viruses isolated early after transmission, we did observe some differences in neutralization sensitivities between early Fiebig stage I/II viruses and Fiebig stage III-V viruses, which may in part contribute to conflicting results reported previously.24,28–30 Our study has some limitations because of the small sample size, and the differences seen here may be due to the features of the viruses from earlier versus later stages of infection or due to novel aspects of subtype CRF01_AE.24 We are now engineering IMCs expressing cognate chronic envelopes to longitudinally measure neutralization profiles of a cohort of subjects who have been intensively studied very early during acute infection.35,36
Considering the RV144 trial results, which are being elaborated in ongoing studies, antibody-mediated protection against HIV-1 acquisition is a likely mechanism, with a component of that reduction attributable to nonneutralizing antibody.6,37–39 Similar findings have been observed in protection from simian immunodeficiency virus acquisition induced by vaccines and passive immunization with mAbs.36,40–42 To best assess the role of neutralizing antibody, future HIV-1 biomedical prevention modalities will continue to require introspection about which antibody neutralization test is used. Understanding the relevance of humoral responses elicited by HIV-1 vaccines may require analysis with viruses being transmitted or found circulating in the population, most of which have a tier 2 phenotype.
In this study, we present a novel panel of full-length CRF01_AE IMC with a reporter gene, generated from different Fiebig acute stages of infection and from chronic infection. These IMCs will be useful for immunological studies, to include different functional humoral responses to HIV-1, and may be used to detect responses to envelopes of differing antibody sensitivities.
The authors are grateful to the RV144 and RV217 study team and volunteers from whom the HIV-1 sequences derived. The authors thank the laboratory and sequence analysis team at Laboratory of Molecular Virology and Pathogenesis for their technical assistance. The authors are thankful to Dr. David Montefiori, Dr. Hongmei Gao, and Ms. Kelli Green for contributing some of the clones generated through the Collaboration of AIDS Vaccine Discovery/Comprehensive Antibody Immune Monitoring Consortium. The authors also appreciate other members of MHRP for their time, efforts, and support.
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