Induction of neutralizing antibodies against tier 2 human immunodeficiency virus 1 in rhesus macaques infected with tier 1B simian/human immunodeficiency virus

We previously developed CCR5-tropic neutralization-resistant simian/human immunodeficiency virus (SHIV) strains and a rhesus macaque model of infection with these SHIVs. We induced the production of neutralizing antibodies (nAbs) against HIV-1 by infecting rhesus macaques with different neutralization-resistant SHIV strains. First, SHIV-MK1 (MK1) (neutralization susceptible, tier 1B) with CCR5 tropism was generated from SHIV-KS661 using CXCR4 as the main co-receptor. nAbs against parental-lineage and heterologous tier 2 viruses were induced by tier 1B virus (MK1) infection of the rhesus macaque MM482. We analyzed viral resistance to neutralization over time in MM482 and observed that the infecting virus mutated from tier 1B to tier 2 at 36 weeks postinfection (wpi). In addition, an analysis of mutations showed that N169D, K187E, S190N, S239, T459N (T459D at 91 wpi), and V842A mutations were present after 36 wpi. This led to the appearance of neutralization-resistant viral clones. In addition, MK1 was passaged in three rhesus macaques to generate neutralization-resistant SHIV-MK38 (MK38) (tier 2). We evaluated nAb production by rhesus macaques infected with SHIV-MK38 #818 (#818) (tier 2), a molecular clone of MK38. Neutralization of the parental lineage was induced earlier than in macaques infected with tier 1B virus, and neutralization activity against heterologous tier 2 virus was beginning to develop. Therefore, CCR5-tropic neutralization-resistant SHIV-infected rhesus macaques may be useful models of anti-HIV-1 nAb production and will facilitate the development of a vaccine that elicits nAbs against HIV-1. Electronic supplementary material The online version of this article (10.1007/s00705-019-04173-5) contains supplementary material, which is available to authorized users.

The rhesus macaque model of simian immunodeficiency virus (SIV) infection is important as an animal model of AIDS for pathogenicity studies and vaccine development. However, the envelope protein (Env) of SIV has a low level of amino acid sequence similarity to that of HIV-1 Handling Editor: Li Wu.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0070 5-019-04173 -5) contains supplementary material, which is available to authorized users. 1 3 [35], and nAbs against the two viruses are not cross-reactive [36]. By contrast, SHIV [37], which is SIV containing the env gene of HIV-1, can be used to evaluate nAbs against the Env protein of HIV-1.
In this study, we evaluated nAb production by rhesus macaques infected with CCR5-tropic tier 1 and tier 2 SHIV. nAbs against tier 2 virus were induced by tier 1B virus infection, and production of nAbs against tier 2 virus began earlier in Tier 2 virus infection. Our findings provide important insights that might be applicable to HIV-1 vaccine development.

Pseudotype viruses
Pseudotype viruses harboring the env gene of MK1, #818, murine leukemia virus (MLV), or clade B panel viruses (NIH AIDS reagent program) were prepared by co-transfecting 293T cells with pSGΔenv and pcDNA3.1 vectors expressing the respective env genes. We obtained pSGΔenv, pcDNA3.1 vectors expressing clade B env, and vectors expressing MLV env from the NIH AIDS reagent program. To construct the pcDNA3.1 vector expressing the rev and env genes of MK1 and #818, approximately 3.0 kb of the region including the rev and env genes of pMK1 [51] and pMK38#818 [52] were amplified by PCR using the primers IFrevF (GCC TTA GGC ATC TCC TAT ) and SHenv7R (GGA GTA TTC ATA TAC TGT CCC). PCR was performed as follows: one cycle of denaturation (94 °C for 2 min), 30 cycles of amplification (98 °C for 10 s, 52 °C for 30 s, and 68 °C for 90 s) and a final extension (68 °C for 10 min) using KOD Plus Neo buffer, 0.2 mM dNTPs, 15 µM primers, 0.02 U of KOD Plus NEO (Toyobo Co., Ltd, Osaka, Japan), and a template. Approximately 5.5 kb of the env-deleted region from pcDNA3.1-SHIVMNA [57] (pcDNA3.1-SHIV-MNA env was generated by InFusion cloning using the pcDNA3.1 vector and SHIV-MNA env PCR product) was amplified by PCR using the primers SHenv7F (GGG ACA GTA TAT GAA TAC TCC) and IFrevR (ATA GGA GAT GCC TAA GGC ). PCR was performed as follows: 1 cycle of denaturation (94 °C, 2 min), 30 cycles of amplification (98 °C for 10 s, 52 °C for 30 s, and 68 °C for 3 min), and a final extension (68 °C, 10 min). The buffer and polymerase were as above. The PCR products were purified using a NucleoSpin Gel and PCR Cleanup Kit (TaKaRa Bio Inc., Shiga, Japan), and env-depleted pcDNA3.1 was reacted with the inserted env DNA. Cloning was conducted using an In-Fusion HD Cloning Kit (TaKaRa), and the resulting plasmid DNA was introduced into Stbl3 cells by electroporation.
Pseudotype viruses harboring the env gene obtained from the plasma of MM482 at weekly intervals after infection were prepared by co-transfecting 293T cells with pSGΔenv and pcDNA3.1 vectors expressing the respective env genes. We produced pSGΔenv in the same manner as above. Viral RNA was extracted from plasma using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. cDNA, including the env gene, was synthesized from the extracted RNA by reverse transcription using random hexamers (Invitrogen, Waltham, MA, USA) and SuperScript IV Reverse Transcriptase (Invitrogen). To construct the pcDNA3.1 vector expressing the rev and env genes of viruses obtained from MM482, at weekly intervals after infection, approximately 3.3 kb of the region including the rev and env genes in the cDNA template was amplified by PCR using the primers SHenv0F (AGA GCA AGA AAT GGA GCC AG) and SHenv8.5R (CCA TAG CCA GCC AAA TGT CT). PCR was performed as follows: 35 cycles of amplification (98 °C for 10 s, 53 °C for 5 s, and 68 °C for 15 s) using KOD One PCR Master Mix (Toyobo), 15 µM primers, and template. Next, approximately 2.9 kb of the region including the rev and env genes in the PCR product was amplified by nested PCR using the primers InsertF3 (TTC ACC GGC TTA GGC ATC TCC TAT GGC AGG AAG AAG CGG AGA ) and InsertR3 (TTG ACC ACT TGC CCC CCA TTT GTC CCT CAC AAG AGA GTG AGC T). PCR was performed as above. The PCR products were purified using a NucleoSpin Gel and PCR Clean-up Kit (TaKaRa) and sequenced directly (Macrogen Japan Corp., Tokyo, Japan). Approximately 5.5 kb of the env-deleted region from pcDNA3.1-SC422661 (obtained from the National Institutes of Health [NIH, Bethesda, MD, USA] AIDS reagent program) were amplified by PCR using the primers VectorF3 (AAT GGG GGG CAA GTG GTC AA) and VectorR3 (AGG AGA TGC CTA AGC CGG TGAA). PCR was performed as follows: 35 cycles of amplification; 98 °C for 10 s, 58 °C for 5 s, and 68 °C for 27 s. The buffer and polymerase were as above. The PCR products were purified, and env-depleted pcDNA3.1 was reacted with the inserted env DNA. Cloning was conducted using an NEBuilder HiFi DNA Assembly Master Mix (NEB Inc., Beverly, MA, USA), and the resulting plasmid DNA was introduced into Stbl3 cells by electroporation.

Neutralization assays
Neutralization assays were performed using various pseudoviruses with pooled plasma from HIV-1-infected patients (ZeptoMetrix, Buffalo, NY, USA) as a positive control. Luciferase activity was measured in TZM-bl cells [58]. Plasma was collected from the infected macaques  50 values were calculated as described previously [39].
As an anti-HIV-1-neutralizing monoclonal antibody, we used KD-247 (which recognizes the epitope GPGR in the V3 region of gp120 and was kindly provided by the Chemo-Sero-Therapeutic Research Institute, Japan). KD-247 was diluted fourfold from 20 to 0.005 µg/mL, and IC 50 values were calculated as previously described [39].

Infection of macaques and antibody production
In MM482 and MM483, the plasma viral RNA level peaked at 10 6 -10 8 copies/mL and was maintained at 10 3 -10 4 copies/mL in MM482. In MM498, MM504, MM481, MM501, and MM502, the plasma viral RNA level peaked at 10 7 -10 8 copies/mL and was maintained at 10 4 -10 7 copies/mL in all of these macaques. In MM596, MM597, and MM599, the plasma viral RNA level peaked at 10 7 -10 8 copies/mL and was maintained at 10 5 -10 6 copies/mL only in MM597. Seven of the ten rhesus macaques developed persistent infections. Many HIV-1-infected patients have a persistent infection with neutralization-resistant virus [39]. To develop a rhesus macaque model of anti-HIV-1 nAb production, we evaluated the neutralization activity and plasma of seven persistently SHIV-infected rhesus macaques (Table 1).

Neutralization against env of parental-lineage virus
Neutralization of parental-lineage virus was evaluated by luciferase assay using human pooled plasma (HPP) from HIV-1-infected patients as the positive control and a pseudovirus containing mouse leukemia virus (MLV) env as the negative control. Potent neutralization of MK1, equal to or higher than that of HPP (ID 50 , 831), was detected in MM482, MM504, MM501, MM502, and MM597 (ID 50 , 10,200, 1,337, 2,679, 831, and 4,587, respectively). Additionally, neutralization activity against #818 higher than that of HPP (ID 50 , 287) was detected in MM504, MM501 and MM597 (ID 50 , 1,357, 907 and 474) ( Table 2). These Table 2 Neutralization activity against parental-lineage virus results imply that nAbs against tier 2 virus are induced in macaques infected with tier 1B virus. Antibodies against MK1 were induced at 6 wpi in MM482 and at 12 wpi in MM501, MM504, and MM597 (Table 3). In contrast, antibodies against #818 were induced at 64 wpi in MM482, 30 wpi in MM504, and at 12 wpi in MM501 and MM597 (Table 3). These results suggest that nAbs against tier 2 virus are induced earlier in macaques infected with tier 2 virus than in those infected with tier 1B virus. Furthermore, the neutralization activity increased over time.

Neutralization against the ENV protein of heterologous viruses
To investigate the ability of the plasma of infected macaques to neutralize a broad spectrum of viruses, we evaluated neutralization activity against heterologous viruses. Potent neutralization of SF162, similar to that of HPP, was detected in all macaques (Table 4). In MM482 and MM597, the neutralization activity of 6535 was similar to that of HPP (ID 50 , 345 and 192, respectively) ( Table 4). In MM482 and MM597, the ID 50 value against REJO4541 was 104 and 103, respectively (Table 4). These results suggest that MM482 and MM597 have broader neutralization activity than the other five macaques, although the neutralization activity of plasma from MM482 and MM597 against tier1B and tier2 viruses was less than that of HPP in all cases (Table 4). In MM482, antibodies against infectious strains and SF162 were induced at 6 wpi (ID 50 , 755) (Table 5A). In MM597, antibodies against infectious strains were induced at 12 wpi (ID 50 , 255), and antibodies were induced against SF162 at 6 wpi (ID 50 , 300) (Table 5B). In MM482, the ID 50 value  (Table 5A and Fig. 1).  (Table 5B and Fig. 1). The neutralization activity of plasma from MM597 against tier 2 viruses at 60 wpi was less than that of plasma from MM482 after 101wpi. Therefore, nAbs against heterologous tier 2 virus are induced by infection with tier 1B virus after 101 wpi. It is also possible that nAbs against heterologous tier 2 viruses begin to be induced after infection with tier 2 virus. These findings suggest that antibody maturation broadens the neutralizing activity over time.

Change from tier 1B to tier 2 virus in an MK1-infected macaque
In the body of HIV-1-infected patients, neutralizing antibodies evolve as the virus mutates [34]. Therefore, neutralizing antibodies may be produced as a tier 1B virus changes to a tier 2 virus in macaques. To confirm this, we analyzed viral resistance to neutralization over time in MM482, which had the highest and broadest neutralization activity. Pseudoviruses with the env gene were collected from plasma during each week of infection and evaluated for neutralization resistance using KD247, #818 (tier 2 virus), and MK 1 (tier 1 B virus). The GPGR epitope of KD 247 was preserved in all viruses from 5 to 115 wpi in the plasma of MM482. Clones 3,4,5,6,8,9, and 10 were prepared from 12 wpi plasma. Clones 6 and 10 contained minor amino acid mutations. All of the clones showed greater resistance to neutralization than the tier 1 B MK 1 virus, but less resistance than that of the tier 2 #818 virus. At 12 wpi, all clones (3, 4, 5, 6, 8, 9, and 10) remained tier 1B (Fig. 2A). Clones 2, 3, 5, 7, and 8 were prepared from 36 wpi plasma and contained minor amino acid mutations. Because clone 7 was more susceptible to neutralization than tier 1B MK 1, it was determined to be a tier 1A virus, and because clone 8 was between MK 1 and #818, it was determined to be a tier 1B virus. Because clones 2, 3, and 5 showed neutralization resistance similar to that of #818, they were determined to be tier 2 viruses (Fig. 2B). Three of five clones were of tier 2 at 36 wpi. Eight, nine and ten clones were prepared from plasma obtained at 46, 70 and 104 wpi, respectively. The neutralization resistance of clones with minor mutations, and of those with consensus sequences, was next evaluated. At 46, 70, and 104 wpi, all clones showed neutralization resistance equivalent to that of #818 and were thus determined to be tier 2 viruses (Fig. 2C,  D, and E). These results suggest that tier 2 virus appeared at 36 wpi, and further that only tier 2 viruses proliferated after 46 wpi in MM482, which was infected with tier 1B MK1.

Mutations related to neutralization resistance and induction of broadly neutralizing antibodies
To identify mutations related to neutralization resistance and induction of broadly neutralizing antibodies, a mutation analysis of env was performed. Direct sequencing showed a consensus sequence lacking minor mutations (Fig. 3). The minor mutations detected in the neutralization-susceptible clones 7 and 8 at 36 wpi were not included. First, common mutations were found from 36 wpi (when neutralizationresistant tier 2 virus appeared) to 115 wpi. These comprised N169D, K187E, S190N in the V2 region, S239 in the C2 region, T459N in the V5 region (T459D at 91 wpi), and V842A in the cytoplasmic tail (Fig. 3, red font). S190N and T459N gained more potential N-linked glycosylation sites compared to MK1. Next, common mutations were found from 91 to 115 wpi; the former was the timepoint at which the maximum neutralizing activity against heterologous viruses was detected. We found the mutations S145N and G149E in the V1 region, D279N in the C2 region, S311P in the V3 region, and I347V and I372V in the C3 region (Fig. 3, blue font). S145N and D279N gained potential N-linked glycosylation sites compared to MK1. These findings imply that these mutations are related to neutralization resistance and induction of broadly neutralizing antibodies.

Discussion
The SHIV strains MK1, MK38, and #818, which were derived from SHIV-89.6, are CCR5-tropic and have different levels of resistance to neutralization (tier 1B and 2) [51,52]. These viruses are genetically similar to SHIV-89.6 P [50], which is widely used in vaccine development. In this study, we developed a rhesus macaque model of induction of anti-HIV-1 nAbs.
In MM597, nAbs against parental-lineage tier 2 viruses were rapidly induced, and nAbs against heterologous tier  (Tables 3 and 5; Fig. 1). In HIV-1-infected patients, self-or type-specific Ab responses develop first, followed by Abs with increased affinity and neutralization activity against autologous viruses [34]. Indeed, neutralization activity against parental-lineage virus increased in rhesus macaques infected with CCR5tropic SHIV. Moreover, in HIV-1-infected patients with nAbs against autologous virus, escape mutants are generated in the virus, and the env sequence diversity increases. Subsequently, the host humoral immune response results in production of nAbs with increased affinity. After a number of years, some patients produce antibodies that target one or more shared epitopes, resulting in cross-reactivity with heterologous strains. This leads to induction of broadly neutralizing antibodies with activity against diverse tier 2 viruses [34]. Therefore, #818-infected rhesus macaques mimic nAb induction in HIV-1-infected patients and may be used to evaluate the induction of tier 2 nAbs.
KS661 was susceptible to neutralization (tier 1B). Induction of Abs in macaques infected with KS661 inhibits viral replication; however, MK38 became resistant to neutralization (tier 2) [52]. MK38 and #818 established persistent infections despite nAb production (Tables 2 and  4), possibly due to the emergence of neutralization-escape mutations or to resistance to nAbs due to the three-dimensional structure of the virus. Indeed, when the co-receptor changes from CXCR4 to CCR5, the resulting decrease in the net positive charge of V3 reduces its surface exposure, resulting in immunological escape from nAbs [52,59].
nAbs against tier 2 parental-lineage and heterologous viruses can be induced by infection with tier 1B virus (Tables 2, 3, and 5; Fig. 1). In HIV-1-infected patients, neutralization results from viral mutations [34]. We analyzed viral resistance to neutralization over time in MM482. In MM482 infected with tier 1B MK 1 virus, the virus mutated from tier 1B to tier 2 at 36 wpi. In MM482,  (Table 3A), and against two of the heterologous tier 2 viruses in the panel after 101 wpi (Table 5A). In MM597 infected with the tier 2 #818 virus, nAbs against three strains of the heterologous tier 2 virus panel were beginning to be detected at 60 wpi, although their ID 50 values were low (Table 5). These results imply that induction of broadly neutralizing antibodies occurs more than 60 weeks after infection with neutralization-resistant virus. In MM482, the S145N, G149E, D279N, S311P, I347V and I372V mutations were detected from 91 wpi to 115 wpi. Since the maximum neutralizing activity against heterologous viruses was detected at this time, six mutations may contribute to the induction of broadly neutralizing antibodies. Analysis of the epitopes targeted by the nAbs induced by MM482 is needed, together with verification that the mutations detected after 91 wpi are important for induction of broadly neutralizing antibodies. It is possible that induction of broadly neutralizing antibodies can be accelerated by a viral antigen with mutations related to neutralization resistance and induction of broadly neutralizing antibodies.
Based on our observations, #818-infected rhesus macaques may be useful models for the induction of tier 2 nAbs. In addition, MK1 (tier 1B)-infected rhesus macaques will enable analysis of the neutralization resistance of viruses that induce tier 2 nAbs and the antigen needed to induce broadly neutralizing antibodies. Finally, these animal models will facilitate the development of HIV-1 vaccines.
Funding This research was supported by Grants-in-Aid for Scientific Research (B) (Grant no. 16H04682) from the Japan Society for the Promotion of Science, and from the Japan Agency for Medical Research and Development (Grant nos. JP18fk0410011, JP18fk0410002, and JP18fk0410013).

Compliance with ethical standards
Conflict of interest The authors have no conflicts of interest to declare.
Ethical approval All animal studies were performed under anesthesia and adhered to protocols approved by the Committee for Experimental Use of Non-Human Primates of the Institute for Frontier Life and Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.