Induction of neutralizing antibodies against tier 2 human immunodeficiency virus 1 in rhesus macaques infected with tier 1B simian/human immunodeficiency virus
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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.
Antiretroviral agents are used against human immunodeficiency virus type 1 (HIV-1), but eliminating latent HIV-1 is difficult [1, 2, 3, 4, 5, 6, 7, 8, 9]. Therefore, suppression and prevention of HIV-1 infection by passive administration of neutralizing antibodies (nAbs) and induction of nAbs by vaccination would be beneficial [10, 11, 12, 13, 14, 15, 16, 17]. Few HIV-1-infected patients (10–30%) produce nAbs, and about 1% of infected people generate highly potent nAbs with broad neutralization coverage of HIV (elite neutralizers) [18, 19]. Due to advances in antigen-specific B-cell isolation techniques, broadly neutralizing monoclonal antibodies have been isolated from HIV-1-infected patients [20, 21, 22, 23]. Passive administration of these nAbs was protective against simian/human immunodeficiency virus (SHIV) in a macaque model [24, 25, 26, 27, 28, 29, 30]. However, inducing potent and broadly reactive nAbs by vaccination is problematic. Although the production of potent nAbs with broad cross-reactivity is related to somatic hypermutation [31, 32, 33, 34], the mechanism of induction is unknown. An animal model in which nAbs are produced would facilitate clarification of the mechanism of induction of nAbs against HIV-1, as well as the development of effective vaccines.
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 , and nAbs against the two viruses are not cross-reactive . By contrast, SHIV , which is SIV containing the env gene of HIV-1, can be used to evaluate nAbs against the Env protein of HIV-1.
Controlling HIV and SIV is difficult, as they use CCR5 as a co-receptor; however, SHIV-89.6P (CXCR4) is easy to control . Seaman et al.  reported that clustering analysis of the patterns of sensitivity defined four subgroups of clinical HIV-1 strains: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization, with the majority of viruses belonging to tier 2. Indeed, the production of antibodies in rhesus macaques suppressed replication of SHIV-KS661(KS661) (CXCR4-tropic, tier 1B) . SHIV-SF162P3 and SHIV-AD8 (tier 2) are used as challenge viruses in vaccine development [33, 41, 42, 43, 44, 45, 46]. We generated several different tier 2 challenge SHIVs to increase the reliability of the research. SHIV-89.6 is frequently used in vaccine studies [47, 48, 49, 50] and thus was selected for this study. First, KS661 (SHIV-89.6 strain), which mainly uses CXCR4 as a co-receptor, was modified to produce SHIV-MK1 (MK1) (tier 1B) and inoculated into rhesus macaques. Next, viruses from the infected macaques were passaged in two macaques, resulting in neutralization-resistant SHIV-MK38 (MK38) (tier 2) . Ishida et al.  produced the MK38 molecular clone SHIV-MK38 #818 (#818) (tier 2).
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.
Materials and methods
HEK293T (293T) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; JR Scientific Inc., Woodland, CA, USA). TZM-bl cells were cultured in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM sodium pyruvate (MP Biomedicals Inc., Santa Ana, CA, USA) and 4 mM L-glutamine (Fujifilm Wako Pure Chemical Corporation). Cells were harvested and passaged using trypsin/ethylenediaminetetraacetic acid solution (Nacalai Tesque, Kyoto, Japan) and were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Viruses and animal experiments
Plasma analyzed for neutralization activity (Matsuda et al., 2010, Virology. Ishida et al., 2015, JGV)
Peak of Plasma Viral Load (copies/mL)
Set point of Plasma Viral Load (copies/mL)
CD4 count in Peripheral Blood
MK1 (molecular clone)
transiently decreased and recovered
Matsuda et al., 2010, Virology.
continuous reduction without signs of recovery
#818 (molecular clone)
Ishida et al., 2015, JGV.
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  and pMK38#818  were amplified by PCR using the primers IFrevF (GCCTTAGGCATCTCCTAT) and SHenv7R (GGAGTATTCATATACTGTCCC). 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  (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 (GGGACAGTATATGAATACTCC) and IFrevR (ATAGGAGATGCCTAAGGC). 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 Clean-up 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 (AGAGCAAGAAATGGAGCCAG) and SHenv8.5R (CCATAGCCAGCCAAATGTCT). 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 (TTCACCGGCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGA) and InsertR3 (TTGACCACTTGCCCCCCATTTGTCCCTCACAAGAGAGTGAGCT). 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 (AATGGGGGGCAAGTGGTCAA) and VectorR3 (AGGAGATGCCTAAGCCGGTGAA). 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 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 . Plasma was collected from the infected macaques [51, 52] and serially diluted, and the 50% inhibitory dilution of the plasma (ID50) was determined with the infectivity of wells lacking plasma defined as 100%. A high ID50 value thus indicates potent inhibition. Plasma from infected macaques was inactivated at 56 °C for 60 min and centrifuged at 11,000 rpm for 10 min. The pooled plasma of HIV-1-infected individuals and infected macaques was diluted in fourfold steps from 1:50 to 1:204,800 and pre-incubated with virus (100 TCID50) at 37 °C for 60 min. Next, 5,000 TZM-bl cells were cultured with the pre-incubated mixture in the presence of 5 mg of DEAE dextran/mL at 37 °C for 48 h. To measure luciferase activity, 50 of µL cell lysate solution (Toyo B-Net, Tokyo, Japan) was added to each well and the plate was agitated for 15 min. An aliquot of 30 µL of lysate was transferred to a Nunc F96 MicroWell white plate (Thermo Fisher Scientific, Waltham, MA, USA), and the luminescent substrate (50 µL) was added to each well. Luciferase activity was calculated with Mikrowin and a TriStar LB 941 reader (Berthold Technologies, Bad Wildbad, Germany). ID50 values were calculated as described previously .
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 IC50 values were calculated as previously described .
Infection of macaques and antibody production
In MM482 and MM483, the plasma viral RNA level peaked at 106–108 copies/mL and was maintained at 103–104 copies/mL in MM482. In MM498, MM504, MM481, MM501, and MM502, the plasma viral RNA level peaked at 107–108 copies/mL and was maintained at 104–107 copies/mL in all of these macaques. In MM596, MM597, and MM599, the plasma viral RNA level peaked at 107–108 copies/mL and was maintained at 105–106 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 . 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 activity against parental-lineage virus
Neutralization activity and breadth against parental-lineage virus over time
Neutralization against the ENV protein of heterologous viruses
Neutralization activity against heterologous viruses
Neutralization activity and breadth against heterologous viruses over time
Change from tier 1B to tier 2 virus in an MK1-infected macaque
Mutations related to neutralization resistance and induction of broadly neutralizing antibodies
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 , 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 2 viruses were beginning to be induced (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 . Indeed, neutralization activity against parental-lineage virus increased in rhesus macaques infected with CCR5-tropic 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 . 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) . 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].
In MM482, the N169D, K187E, S190N, S239, T459N (T459D at 91 wpi), and V842A mutations were detected at 36 wpi. Because the tier 2 #818 virus has three mutations in the V2 region (N169D, K187E, and S190N) (Supplemental Figure), the above-mentioned six mutations likely contribute to neutralization resistance.
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 . 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, nAbs against the tier 2 #818 virus were detected at 64 wpi (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 ID50 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.
We thank the NIH AIDS Reagent Program for providing a panel of reference subtype B env clones.
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.
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 Medical Sciences, Kyoto University, Kyoto, Japan. Macaques were housed in a biosafety level 3 facility in which all of the procedures were performed.
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