An effective HIV-1 vaccine will most likely require the ability to induce broadly neutralizing antibodies (bnAbs) against diverse globally circulating HIV-1 strains. During natural HIV-1 infection, about 20% of HIV-1-infected individuals develop neutralization activity with significant breadth after 3–5 years of infection [1–5]. A large number of bnAbs targeting five major conserved epitopes on HIV-1 envelope glycoprotein have been identified from a small number of HIV-1-infected individuals and have been fully characterized for their crystal structures and target epitopes [6,7]. Infusion of bnAbs can protect repeated simian/human immunodeficiency virus (SHIV) challenges in macaques  and HIV-1 challenges in humanized mice [9,10]. These results raise the hope that bnAbs can protect acquisition of infection if they are elicited by vaccination. Although no vaccines have been able to elicit bnAbs in humans or nonhuman primates (NHPs), nAbs neutralizing autologous tier 2 viruses could be detected in NHPs [11,12] and increased neutralization breadth was induced by the germline targeting approach in which the reverted bnAb germline was knocked in mice .
The only animal model to evaluate protection efficacy of a nAb-based vaccine is NHP by challenging with SHIVs, in which the env gene from the simian immunodeficiency virus (SIV) is replaced by the env gene from HIV-1. SHIV-infected NHPs are generally not maintained more than 2–3 years after infection when planned experiments are accomplished. As broad neutralization activity is generally detected after 3–5 years of infection in humans [14–17], bnAbs do not have enough time to develop in those macaques. This may explain why only a few macaques were found to have relatively broad neutralization activity within 1–2 years of infection [18–21]. In this study, we have followed macaques infected with different SHIVs for up to 6 years and found broad neutralization activities in two macaques after 5–6 years of infection. Furthermore, their neutralization specificities (CD4bs, V3 and V2) were similar to those found in HIV-1 infected humans. More detailed characterization of bnAbs in those macaques will be highly informative for vaccine design to elicit broadly reactive vaccines and appropriacy of the SHIV/NHP model to evaluate bnAb-based vaccines.
Simian/human immunodeficiency virus infection in nonhuman primate
Chinese rhesus (Rh) macaques were challenged with SHIV1157ipd3N4, SHIVSF162P3 or SHIVCHN19P4 intravenously at various doses (5–500 TCID50) or at multiple low-dose method intrarectally up to 10 times (10–50 TCID50). Persistent infection was determined by measuring viral loads in plasma using a quantitative real-time-PCR as previously described . All macaques were cared for in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Science and with the recommendations of the Weatherall report (the use of NHPs in research). All animals tested negative for simian type D retroviruses, simian T-cell leukemia virus-1, and SIV before the start of each study.
Neutralizing titers of plasmas were assessed by single-round infection of TZM-bl with envelope (Env) pseudoviruses as described previously . Briefly, inactivated plasma samples were prepared at three-fold serial dilutions starting at 1 : 30. The diluted samples were incubated with Env pseudoviruses (100 TCID50) for 1 h at 37 °C and then used to infect TZM-bl cells. The luciferase activity was measured after 48 h. The definition of 50% inhibitory dose (ID50) reported as plasma reciprocal dilution was the sample dilution at which relative luminescence units (RLU) were reduced by 50% compared with RLU in virus control wells after subtraction of background RLU in cell control wells. A response was considered positive for neutralization if the ID50 titer was more than 1 : 30.
Neutralization specificity analysis
Mutations that affect bnAb binding to V2 (N160K), V3 (N332A) and CD4bs (N279A, N280D and V295N) regions were introduced into the Ce1176 env clone using the QuikChange mutagenesis kit (Stratagene, La Jolla, California, USA). The identity of all mutants was confirmed by sequencing and the neutralization susceptibility of pseudotyped viruses bearing mutant Env was confirmed by TZM-bl assay against a panel of bnAbs as previously described . CAP45.2.00.G3 env mutants containing mutations at V2 (N160A, T162A, R166A, D167N, K169V and K169E), CD4bs (T236K and N276D) and gp41 (N616A, N637A and N674A) regions were generated from a previous study . Pseudoviruses were produced by transfection of 293T cells with an env-deficient genomic back-bone plasmid (pSG3ΔEnv) and each env mutant. Potential epitopes were determined by resistance of mutants to plasma from G1015R or G1020R collected at week 321.
To test whether breadth and potency of the neutralization responses increased over time, we used a mixed-effect generalized linear model (GLM) with the neutralization data as dependent variable (both magnitude and breadth), SHIV stains and time as independent variables, as well as Envs and animals as random effects. We tested for possible interactions between all independent variables, and then, in a separate model, we used all independent variables additively and then stepwise removed nonsignificant variables until we found the best predictive model. All analyses were performed in R  using the lme4 package  and all P values were obtained through the analysis of variance chi-square test.
Persistent simian/human immunodeficiency virus infection in Chinese rhesus macaques
Previous studies showed that susceptibility to SIV infection and death rates were lower in Chinese Rh macaques compared with Indian Rh macaques [31,32]. To determine if SHIV can also establish long-term infection with relatively high viral loads, which are generally required for inducing bnAbs in NHPs  and humans [34,35], we studied the persistent infection among 40 SHIV-infected macaques established from our previous study . Twenty macaques were infected with SHIVSF162P3 and the other 20 macaques were infected with SHIV1157ipd3N4. Each virus was given intravenously or intrarectally at various doses. Nine intravenously infected macaques did not establish infection due to low doses. Among 31 infected macaques, 15 were infected with SHIVSF162P3 (10 intrarectal infection and five intravenous infection), whereas 16 were infected with SHIV1157ipd3N4 (10 intrarectal infection and six intravenous infection) (Fig. 1). All infected macaques had high peak viremia during the acute infection stage. Viral loads decreased to the baseline around 30 weeks after infection, but rebounded to various levels after 1 year of infection. Viral loads were measured every 1 or 2 years during the next 170 weeks. Throughout the follow-up period, no macaques died of AIDS-related symptoms. All monkeys were euthanized at week 217 except G1015R and G1020R (infected with SHIV1157ipd3N4), which maintained high viral loads for next 104 weeks.
Broad neutralization activity in simian/human immunodeficiency virus-infected monkeys
Archived longitudinal plasma samples were available only from six macaques. Of these, G1010V, G1015R and G1020R were infected with SHIV1157ipd3N4, whereas G0802V, G0815R and G0821R were infected with SHIVSF162P3. Longitudinal plasmas were available from these animals at various time points: weeks 54–217 for G0802V, G0815R, G0821R and G1010V, but up to 321 weeks for G1015R and G1020R. Baseline plasma samples were available from all six macaques. To determine the plasma neutralization breadth from those six monkeys, we measured their neutralizing activity against a panel of 17 tier 2 viruses (difficult-to-neutralize), two tier 1 viruses (easy-to-neutralize) and autologous viruses (SF162.LS and 1157ipd3N4) (Fig. 2). Autologous nAbs were detectable at week 27 and maintained at high titers throughout the follow-up period in all but G0815R. They also neutralized tier 1 virus MW965.26 as potently as autologous viruses, but neutralized 92RW020.2 less potently. Although nAbs were detected much later in G0815R than in other macaques, the plasma neutralized all four viruses similarly as those from other monkeys by week 217.
To determine neutralization breadth and potency of plasma from these monkeys against tier 2 viruses, we performed neutralization assays with 17 tier 2 pseudoviruses that represent multiple subtypes (A, B, C and G), circulating recombinant forms (CRF01, CRF07) and a unique subtype AC recombinant [36–40]. This panel included all 12 viruses in the global panel that was recommended to evaluate the neutralization breadth of anti-HIV-1 sera . Plasmas from weeks 27–54 neutralized only 1–4 viruses (Fig. 2). These low titer neutralization activities were mainly found against 25710 and ZM1197M.PB7. Neutralization activities became more frequently detectable by year 4 (week 217) in three of four monkeys (G0815R, G1015R and G1020R) and the neutralization titers were slightly higher than those from year 1. They neutralized an average of 5.5 (1–8) viruses (Table 1). Plasmas after year 5 (weeks 261–321) were only collected from G1015R and G1020R. They all neutralized more viruses than plasmas from week 217. By week 321, an average of 11.5 (11–12) tier 2 viruses were neutralized. The later plasmas from G1020R had the broadest neutralization spectrum: 14 (83%) and 12 (71%) viruses for plasmas from week 293 and week 321, respectively.
To determine whether breadth of the neutralization activity significantly increased over time, we ran a mixed-effect GLM on the data from the four monkeys (G0815R, G0821R, G1015R and G1020R) that had three common time points (weeks 27, 54 and 217). We found a strong interaction effect between SHIVs as well as between the subtypes and tier types of the Env-pseudoviruses (P = 8 × 10−5 and 0.0017 on tier 1 and 2 viruses, respectively), indicating that responses were significantly different across different SHIVs, subtypes and tiers. We then restricted our analyses to tier 2 only viruses and found that the magnitude of neutralization responses did not significantly increase at week 54 compared with week 27 (P = 0.21), but they did by week 217 (P = 4.3 × 10−9) even though by only a 50% increase on average. Responses were 1.3 times higher in SHIV1157ipd3N4 compared with SHIVSF162P3. When looking at breadth, the chance of getting positive responses did not statistically significantly change between week 27 and week 54 (P = 0.4), but by week 217 the chance of getting positive responses was over 12-fold higher compared with week 27 (P = 0.015). The chance of getting positive responses in SHIV1157ipd3N4 was 2.7 times higher compared with SHIVSF162P3, but this difference was NS (P = 0.09).
Longitudinal samples were collected from G1015R and G1020R over a time spanning 321 weeks since infection. This allowed us to test whether the breadth of the neutralization activity against tier 2 viruses significantly increased over 6 years of infection. We ran the same model on the two monkeys only, using data from weeks 27, 54, 217, 261, 293 and 321. The chance of getting a positive response at week 217 was significantly higher than at weeks 27 and 54 (P = 0.001 and 0.089, respectively), but equivalent to the chance of getting a positive response at week 261 (P = 0.99). At week 293, there was a 5.5-fold increase in the chance of getting a positive response compared with week 217 (P = 0.012), and at week 321, there was a 4.5-fold increase in the chance of getting a positive response compared with week 217 (P = 0.027). No differences between weeks 293 and 321 were observed. This indicated that the neutralization breadth significantly increased by week 293, but plateaued by week 321 (Fig. 2).
To determine whether antibodies in the plasmas from the two macaques that developed broad neutralization activities could bind the Env glycoprotein of the challenging virus better over time, we measured their binding ability by ELISA. The binding abilities of plasmas to SHIV1157ipd3N4 Env generally increased over time in G1015R (Supplementary Fig. 1A, http://links.lww.com/QAD/B204). However, all plasmas after week 27 in G1020R bound SHIV1157ipd3N4 Env at similar levels, with the exception of the week 321 plasma that showed weaker binding at higher dilutions (Supplementary Fig. 1B, http://links.lww.com/QAD/B204).
Development of distinct neutralization patterns in monkeys infected with the same virus
Both G1015R and G1020R developed broad neutralization activities against 17 tier 2 viruses after 5–6 years of infection (Table 1). However, the neutralization patterns were strikingly different between them (Fig. 2). Plasmas from both macaques at those time points could neutralize seven viruses (CE0217, BJOX2000, CE1176, X2278, 25710, ZM197M.PB7, 16936–2.21 and SC422661.8) similarly and did not neutralize CNE8 and CNE55, except one sample at week 293 from G1020R. Neutralization activities against X1632, CH119, TRO11, 246F3 and 398F1 were developed in G1020R at weeks 293 and 321, but neutralization activities against only X1632 and 398F1 were detected in G1015R at week 321. On the other hand, G1015R developed relatively strong neutralization activity against WITO4160.33 as early as week 54 and CAP45.2.00.G3 at week 217, and nAb titers were maintained at the similar levels throughout the follow-up period. However, G1020R never developed neutralization activity against WITO4160.33 and CAP45.2.00.G3. These distinct neutralization profiles in G1015R and G1020R suggested that different nAbs were elicited even though both macaques were infected by the same virus through the same infection route.
Disparate HIV-1 neutralization breadth among different SHIV-infected monkeys
Single time point plasma samples were available from additional four SHIV1157ipd3N4-infected macaques, four SHIVSF162P3-infected macaques and five SHIVCHN19P4-infected macaques after 6 years of infection. To determine if macaques infected with different SHIVs would also induce similar broad neutralization activity after 6 years of infection, we analyzed plasma samples from those monkeys against the recommended global panel of 12 tier 2 pseudoviruses (Supplementary Fig. 2, http://links.lww.com/QAD/B204). Plasmas from SHIV1157ipd3N4-infected macaques neutralized an average of 6.3 (1–9) viruses (Fig. 3). This was significantly higher than plasmas from SHIVSF162P3-infected macaques or SHIVCHN19P4-infected macaques, which only neutralized 1.4 (0–3) and 1.6 (0–3) viruses on average, respectively (P = 0.017 and P = 0.035, respectively; Student's t test). Significantly, plasmas from four SHIV1157ipd3N4-infected macaques by intrarectally neutralized 7–9 viruses, whereas two SHIV1157ipd3N4-infected macaques by intravenously only neutralized one or three viruses. However, all SHIVSF162P3-infected macaques by intrarectally only neutralize as many as three viruses. These results show that different SHIVs and infection routes can have significant impacts on induction of bnAbs as previously reported , and SHIV1157ipd3N4 was superior to SHIVSF162P3 and SHIVCHN19P4 for induction of broad neutralization responses.
Determination of specificities of broadly neutralization activities
We next sought to determine neutralization specificities of nAbs in SHIV-infected macaques. As broad neutralization activities were elicited in G015R and G1020R, to determine if three major bnAb epitopes (V2, V3 and CD4bs) were targeted, we mapped their neutralization specificities using two sets of HIV-1 env mutants. Ce1176 could be neutralized by plasmas from both G1015R and G1020R (Fig. 2). Thus, we first determined their neutralization activity against wild type (wt) Ce1176 and five mutants with mutations in V2 (N160K), V3 (N332A) and CD4bs (N279A, N280D and V295N) regions (Fig. 4a and b). Both plasmas could not neutralize N160K and N332A mutants, suggesting that the V2 and V3 regions were targeted by nAbs in both macaques. For three CD4bs mutants, G1015R and G1020R plasmas did not neutralize mutant V295N but more potently neutralized mutant N280D. G1015R and G1020R plasmas neutralized the mutant N279A differently: the G1020R plasma only weakly neutralized mutant N279A, whereas the neutralization titer by G1015R plasma was marginally reduced. These results suggest that nAbs targeting CD4bs were developed in both macaques, but in slightly different manners.
CAP45.2.00.G3 was neutralized by only G1015R plasma (Fig. 2). To further confirm the neutralization specificities identified with the Ce1176 mutants, we tested G1015R plasma against a set of 11 CAP45.2.00.G3 mutants . Among six V2 mutants, three (N160A, T162A and K169E) were fully resistant to neutralization by the G1015R plasma (Fig. 4c). Although the R166A and K169V mutants were only slightly more resistant to the G1015R plasma, mutant D167N was more sensitive to the G1015R plasma than the wt virus. The complete resistance of N160K in Ce1176 and N160A in CAP45.2.00.G3 to neutralization by the G1015R plasma confirmed that nAbs targeting the V2 region were present in G1015R. Between two CD4bs mutants which were different from those in Ce1176, mutant T236K was slightly more sensitive to the G1015R plasma than the wt virus, whereas mutant N276D was 44% more resistant than the wt virus. This suggested that CD4bs was likely targeted in G1015R, but not as potently as in G1020R. No significant differences in neutralization susceptibility were observed between gp41 mutants (N616A, N637A and N674A) and the wt virus, suggesting that those sites were not targeted by nAbs in G1015R.
Broad neutralization activities were also detected in G0021R and G0022R (Fig. 3 and Supplementary Fig. 2, http://links.lww.com/QAD/B204). Plasmas from both macaques neutralized Ce1176 but not CAP45.2.00.G3. Thus, we used the Ce1176 mutants to investigate the possible targets of nAbs in both macaques. The G0021R plasma did not neutralize N160K, N280D, V295N and N332A mutants, whereas it more potently neutralized the N279A mutant (Supplementary Fig. 3A, http://links.lww.com/QAD/B204), suggesting that the V2, CD4bs and V3 regions were targeted by nAbs in G0021R, similar to what were observed in G1020R (Fig. 4a). In contrast, the G0022R plasma neutralized all five mutants similarly; the variations in neutralization potency were less than two folds compared with the wt Ce1176 (Supplementary Fig. 3B, http://links.lww.com/QAD/B204). These results suggested that nAbs in G0022R might target epitopes not in the V2, CD4bs and V3 regions.
NHPs are the only animal model to evaluate if protective immunity against HIV-1 is elicited by nAb-based vaccines. Thus, understanding if and how bnAbs are elicited during SHIV infection in NHPs can reveal critical clues for the design of more effective vaccines. By studying a group of long-term SHIV-infected macaques, we found that, like in HIV-1-infected humans, SHIV-infected macaques developed broad neutralization activities after years of infection, and that multiple bnAb epitopes in V2, V3 and CD4bs regions were targeted. These findings can have significant implications in design and evaluation of future HIV-1 Env vaccines in NHPs.
Few SHIV-infected NHPs have been maintained for more than 3 years [19–21]. Thus, a group of long-term SHIV-infected NHPs in this study gave us an opportunity to study how broad neutralization activities developed in these monkeys. Broad neutralization activities were rarely detected within 2 years of infection. By year 4, broad neutralization activities were significantly increased. In two monkeys that were followed for 6 years, plasmas neutralized 68% of tier 2 viruses by year 6. These observations demonstrated that, like in HIV-1-infected humans, SHIV-infected NHPs could also develop broad neutralization activities after over 5–6 years of infection. This indicates that bnAbs in SHIV-infected NHPs may also require a lengthy maturation process as in HIV-1-infected humans. As elicitation of bnAbs is most likely required for a successful AIDS vaccine, the key issue for future immunogen design will be to shorten the bnAb maturation time by optimizing the immunization scheme.
Analysis of a group of macaques that were infected for 6 years showed that different SHIVs had various abilities to elicit broad neutralization activities. SHIV1157ipd3N4-infected macaques had significantly broader neutralization activities than SHIVSF162P3-infected or SHIVCHN19P4-infected macaques. Among six SHIV1157ipd3N4-infected macaques, four intrarectally infected macaques had broader neutralization activities than the other two intravenously infected macaques. This is an encouraging observation that given enough time, infection of NHPs by some SHIVs through an appropriate route can likely induce broad neutralization activities. On the contrary, all four macaques infected with SHIVSF162P3 intrarectally did not elicit broad neutralization activities. Infection of five macaques by SHIVCHN19P4 also failed to induce broad neutralization activities. This is in line with the observation in that only low-tier neutralization activity against a tier 1 virus was detected in SHIVDH12-infected macaques after 5 years of infection . Although the number of macaques was relatively small, these results indicate that different SHIV strains and infection routes can have a significant impact on development of bnAbs after infection.
Previous studies showed that broad neutralization activities were only found in a few macaques after screening a large number of monkeys within 1–2 years of infection [19,21]. We found that two out of three SHIV1157ipd3N4-infected macaques were able to develop broad neutralization activities that neutralized at least 65% of tier 2 viruses after 5 years of infection. High viral loads and greater diversity of the env gene sequences may played an important role in development of cross-reactive neutralization in SHIVAD8-infected Rh macaques  as well as in infected humans [34,35]. Thus, high viral loads in late stage of infection that allowed viruses to accumulate more mutations may be key for development of broad neutralization activities in both G1015R and G1020R macaques. One study showed that plasmas from some SHIVSF162P3N-infected Rh macaques could broadly neutralize eight tier 2 viruses by 96 weeks post infection . In our study, however, plasmas from SHIVSF162P3-infected macaques only neutralized 1–6 tier 2 viruses. This was probably due to the use of different challenging SHIV strains (SHIVSF162P3N versus SHIVSF162P3) and monkey species (Indian Rh macaques versus Chinese Rh macaques) in two studies [41,42].
Plasmas from G1015R and G1020R showed distinct neutralization profiles with a panel of 17 tier 2 viruses. As both macaques were infected intrarectally with the same SHIV1157ipd3N4 stock, these results suggest that hosts with different genetic backgrounds might have an impact on development of bnAb specificities. Although this observation warrants further validation with more monkeys, this observation may have important implications in future vaccine design.
Potential epitopes targeted by antibodies from macaques with broad neutralization activities was mapped to the gp120 N332 glycan site in one monkey infected with SHIVAD8. In this study, we found that neutralization specificities for three common bnAb epitopes (V2, V3 and CD4bs) were all likely targeted in three SHIV1157ipd3N4-infected macaques (G1015R, G1020R and G0021R). This is very encouraging as bnAbs similar to those targeting V1V2, V3 and CD4bs by human bnAbs are likely elicited by SHIV in NHPs. This raises the hope that a better designed vaccine approach can elicit broad neutralization activities in NHPs. These results demonstrate that SHIV1157ipd3N4-infected Chinese Rh macaques can serve as a proper model to study bnAb maturation.
The number of monkeys in this study is relatively small, and the neutralization breadth was not the initial goal of the study. Thus, only limited blood samples were preserved for analysis neutralization breadth. Therefore, a well designed study with numbers of SHIV1157ipd3N4-infected monkeys better powered to confirm our observations is desirable, as it would help to elucidate mechanisms of bnAb maturation in NHPs. However, two monkeys were still alive after 6 years of infection and blood samples were regularly collected. More importantly, peripheral blood mononuclear cells from both monkeys have been cryopreserved. This will allow us to isolate monoclonal bnAbs from these monkeys to more precisely determine whether the evolution pathways, usages of B cell germlines and specificities of bnAbs in NHPs are similar to those in humans. Knowledge learned from these studies will have important implications for better utilization of NHP models for AIDS vaccine development.
We thank David C. Montefiori and Celia C. LaBranche for the global panel of 12 HIV-1 env clones; Penny Moore and Lynn Morris for CAP45.2.00.G3 env mutants; Ruth Ruprecht for NL-LucR.1157ipd3N4 clone; and National Institutes for Food and Drug Control for the tier 1 and 2 env clones.
The current work was supported by the National Natural Science Foundation of China (grant no. 31670162), Program for JLU Science and Technology Innovative Research Team (grant no. 2017TD-05) and CAMS Innovation Fund for Medical Sciences (grant no. 2016-I2M-1–019).
Author contributions: F.G. and X.Y. conceived the study; N.G., W.W., C.W., T.G. and R.G. performed experiments; N.G., W.W., C.W., B.Y., X.Y., C.Q. and F.G. analyzed data; E.E.G. performed statistical analysis; Z.C. generated the SHIV clone; S.T. and S.H. generated the env mutants; N.G., W.W., C.W., X.Y., E.E.G., S.H. and F.G. wrote and edited the article.
Conflicts of interest
There are no conflicts of interest.
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* These two authors contributed equally to this work.
† These three authors contributed equally to this work.