Most rhesus macaques infected with the CCR5-tropic SHIV_AD8 generate cross-reactive antibodies that neutralize multiple HIV-1 strains

Abstract

The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIV_AD8-EO) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1_DH12 strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIV_AD8 stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIV_AD8-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti–HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41–51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIV_AD8 macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains.

A major challenge in HIV vaccine research has been the development of immunogens capable of eliciting potent, broadly acting, neutralizing antibodies (NAbs). It is now appreciated that 10–30% of HIV-1–infected individuals produce cross-reactive NAbs of significant breadth (1–6). Less than 1% of such persons, so-called “elite neutralizers,” produce potent cross-clade–neutralizing activity, but only 2–3 y after virus acquisition (6). Although the emergence of broadly reacting anti–HIV-1 NAbs in elite neutralizers has been associated with multiple rounds of somatic hypermutation (7), little is known about vaccine strategies able to elicit such antibodies. Longitudinal studies of HIV-1– infected persons have suggested that set-point plasma virus loads, CD4⁺ T-cell levels, duration of the infection, or antibody-binding avidity may contribute to the development of cross-reacting NAbs (5, 8, 9). It is also possible that the induction of such antibodies depends on unique gp120 epitopes associated with specific HIV-1 strains and individual B-cell repertoires or is simply a random process (10, 11). A nonhuman primate model capable of generating cross-reactive anti–HIV-1–neutralizing activity could provide answers to some of these questions and contribute to the development of an effective prophylactic vaccine. In this regard, we recently reported that one rhesus monkey, inoculated with an uncloned preparation of the R5-tropic SHIV_AD8, developed broad, potent, and glycan-specific NAbs with cross-reacting activity against virus isolates from different HIV-1 clades similar to that described for “elite” HIV-1 neutralizers (12).

In this study, we describe the construction of a pathogenic SHIV_AD8 molecular clone (SHIV_AD8-EO). During its characterization, we discovered that eight of eight SHIV_AD8-EO–infected animals generated cross-reactive NAbs directed against a SHIV carrying an envelope glycoprotein derived from a different HIV-1 isolate (namely HIV-1_DH12). Because this result suggested that a majority of monkeys infected with the SHIV_AD8-EO family of viruses might be able to generate NAbs against heterologous HIV-1 isolates, plasma samples collected from a cohort of 11 rhesus macaques, infected with either uncloned SHIV_AD8 swarm stocks or SHIV_AD8 molecular clones, were tested for their capacity to neutralize a panel of clade A, B, and C HIV-1 isolates. All of the plasmas from this group of 11 animals neutralized several tier 1A and 1B clade B HIV-1 strains. Three of the 11 macaques also generated >1:100 IC₅₀ neutralization titers against some tier 2 clade B HIV-1 isolates, and one of the three produced significant NAb titers against some clade A and C isolates. Taken together, these findings indicate that SHIV_AD8 is uniquely immunogenic during infections of rhesus monkeys and may be a particularly useful reagent for identifying viral determinants that drive B-cell maturation resulting in cross-reactive anti–HIV-1 NAbs.

Results

Infection of Rhesus Monkeys with the SHIV_AD8-EO Molecular Clone.

Although we had previously reported the construction and characterization of the R5-tropic SHIV_AD8 and had prepared uncloned SHIV_AD8 swarm stocks as challenge viruses for vaccine experiments (13, 14), we had not obtained a pathogenic SHIV_AD8 molecular clone, capable of durably maintaining chronic virus infection and inducing clinical immunodeficiency in inoculated animals. This was accomplished, as described in Materials and Methods, by amplifying SHIV_AD8 vpu and env genes from the plasmas of several SHIV_AD8–infected monkeys and inserting individual amplicons into the genetic background of SIVmac239 (Figs. S1 and S2). One of the resulting clones (SHIV_AD8-EO) exhibiting robust replication kinetics in cultured rhesus peripheral blood mononuclear cells (PBMC) was inoculated into 11 animals: 4 by the i.v. (5,000 or 500 TCID₅₀) and 7 by the intrarectal (5,000 or 1,000 TCID₅₀) routes. Similar to uncloned SHIV_AD8 derivatives (13, 14), the levels of set point viremia in macaques infected with the SHIV_AD8-EO molecular clone varied widely (10² to >10⁵ RNA copies/mL) (Fig. 1A). Memory CD4⁺ T cells at an effector site [recovered by bronchoalveolar lavage (BAL)] declined markedly in all of the monkeys during the acute phase of the SHIV_AD8-EO infection (Fig. 1B), and a gradual loss of total circulating CD4⁺ T cells occurred in most animals (Fig. 1C). Monkey DC0L was euthanized at week 95 post infection (PI) with clinical symptoms of immunodeficiency following protracted anorexia and diarrhea that was accompanied by marked weight loss. Macaque DC6W also experienced marked weight loss and was euthanized at week 111 with a gastric lymphoma. Macaque CD8T was euthanized at week 117 with multiple intra-abdominal lymphomas.

Fig. 1.

Virus replication and CD4⁺ T-cell dynamics in rhesus macaques inoculated i.v. or intrarectally with the molecularly cloned SHIV_AD8-EO. The levels of plasma viremia (A), percentage of BAL fluid CD4⁺ T cells (B), and absolute numbers of circulating CD4⁺ T cells (C) are shown.

Neutralizing Antibodies Generated in SHIV_AD8-Infected Macaques.

We recently reported that only 3 of 19 macaques inoculated with SHIV_AD8 swarm viruses developed sustained levels of autologous Nabs (13). In the current study, autologous NAbs, developing in eight of the macaques infected with the SHIV_AD8-EO molecular clone were initially assessed using plasma samples diluted 1:20. As shown in Fig. 2A, five of the eight animals produced autologous neutralizing activity (>50% neutralization). In four of these macaques (DC6W, DCF1, FZH, and JG7), autologous NAbs became measurable between weeks 20 and 40 PI, but were delayed until week 74 in the fifth (DC8T) animal. Individual samples from three of these monkeys (DC6W, DCF1, and JG7) had autologous neutralization IC₅₀ titers ranging from 1:148 to 1:161.

Fig. 2.

Autologous and cross-reacting NAbs produced by animals infected with the molecularly cloned SHIV_AD8-EO. Plasma samples from the indicated SHIV_AD8-EO–infected macaques were collected at different times post infection, diluted 1:20, and assayed for neutralizing activity against the autologous SHIV_AD8 (A) or the heterologous SHIV_DH12 (B). The dashed line in each panel at 50% neutralization represents the threshold of virus suppression in the TZM-bl cell assay. The IC₅₀ neutralization titers determined for plasmas collected from macaques DC6W (week 52), DCF1 (week 40), and JG7 (week32) were determined by reciprocal dilution and assay in TZM-bl cells. Asterisks indicate NAbs detected at week 95. nd: not done.

In an earlier study, we reported that one rhesus monkey inoculated with uncloned SHIV_AD8 developed extraordinarily broad, cross-clade, and high-titered NAbs similar to that described for HIV-1 “elite neutralizers” (12). This potent neutralizing activity targeted the gp120 N332 glycan. Because we were curious whether animals infected with the SHIV_AD8-EO molecular clone might also produce antibodies able to neutralize a heterologous HIV-1 isolate, plasma samples (1:20 dilution) from the same eight SHIV_AD8-EO–infected monkeys were tested for their capacity to neutralize pseudovirions bearing the CXCR4 (X4)-tropic SHIV_DH12-CL7 envelope glycoprotein, originally derived from the HIV-1_DH12 isolate (15–17). The HIV-1–derived env genes present in SHIV_AD8-EO and SHIV_DH12-CL7 are 90% and 84% identical at the level of nucleotide and amino acid sequences, respectively. As shown in Fig. 2B, all eight plasmas, including samples from animals (DC0L, DC7W, and DCV9) with no or extremely low autologous NAbs, exhibited neutralizing activity against SHIV_DH12.

We next determined the cross-reactive IC₅₀ NAb titers directed against SHIV_DH12 present in the plasmas of a different cohort of 11 macaques, which had been inoculated with molecularly cloned SHIV_AD8 or recently described SHIV_AD8 swarm stocks (13) (Fig. 3A). Only 4 (macaques DC6W, DCF1, DA55, DA70) of these 11 SHIV_AD8–infected animals had developed autologous NAbs. Nonetheless, the plasmas from all 11 SHIV_AD8–infected animals generated anti-SHIV_DH12 neutralizing IC₅₀ titers (ranging from 1:236 to 1:3,040).

Fig. 3.

Macaques infected with uncloned or molecularly cloned SHIV_AD8 viruses develop cross-reacting NAbs against multiple HIV-1 strains. IC₅₀ neutralization titers against the highly sensitive SHIV_DH12 (A), the indicated clade B (B) and clade A and C (C) HIV-1 isolates in plasmas of a cohort of 11 SHIV_AD8–infected animals were determined by reciprocal dilution and assay in TZM-bl cells. Values represent reciprocal serum dilution required to achieve 50% neutralization. The previously reported (12) neutralizing activity of week 100 plasma from animal CE8J is shown at the bottom of each panel. Value between 20 and 99, green; value between 100 and 999, yellow; value ≥1,000, red. Blank cell indicates that value was not determined.

The production of cross-reacting NAbs against SHIV_DH12 by SHIV_AD8–infected monkeys raised the possibility that a similar activity might have also been generated against other HIV-1 isolates. Plasma samples from the same cohort of 11 animals, infected with the molecularly cloned or uncloned SHIV_AD8 viruses, were assessed for their capacity to neutralize a panel of clade A, B, and C HIV-1 strains. As shown in Fig. 3 B and C, plasmas from all of the monkeys possessed neutralizing activity against tier 1A (very high sensitivity) and tier 1B (above-average sensitivity) clade B HIV-1 isolates (18). In addition, three of the macaques (CL5E, DCF1, and DA55) generated >1:100 IC₅₀ NAb titers against some tier 2 (moderate sensitivity) clade B HIV-1 strains (HIV-1_JR-FL, HIV-1₇₆₅₁₅, HIV-1_TRO.11, and HIV-1_CAAN.A2). The plasma from monkey CL5E exhibited the widest breadth, including neutralization activity against clade A and C HIV-1 strains. A neutralization profile for the previously reported (12) “elite neutralizer” macaque CE8J at the time of its euthanasia at week 100 PI is also shown at the bottom of Fig. 3.

In the context of the neutralization phenotypes shown in Fig. 3, SHIV_DH12 ranks with several highly neutralization-sensitive clade B HIV-1 strains. However, when its sensitivity was evaluated against a panel of standardized plasma pools from HIV-1 infected individuals (18), SHIV_DH12 exhibited a tier 2 neutralization phenotype (Table S1).

To ascertain whether the temporal appearance of cross-reacting NAbs directed against the more difficult-to-neutralize tier 2 HIV-1 strains occurred at the same time as neutralizing activity against tier 1 isolates in macaque CL5E (Fig. 3), the IC₅₀ neutralization titers present in plasma samples collected at various times following SHIV_AD8 inoculation of this animal were determined. Surprisingly, significant levels of neutralizing activity became detectable only against the tier 1B HIV-1_SS1196.1 strain by week 13 PI (Fig. 4A). NAbs directed against three tier 2 clade B HIV-1 isolates or clade A and C HIV-1 strains all emerged between weeks 41 and 51 PI. In an independent experiment, cross-reacting NAbs directed against the highly neutralization-sensitive SHIV_DH12 appeared between weeks 7–10 PI in two representative SHIV_AD8–infected monkeys (Fig. 4B). Taken together, these results suggest that SHIV_AD8–infected macaques may produce cross-reactive neutralization activity in one or two phases of the infection: (i) at early times (weeks 7–13 PI), a majority of animals may generate high titers against relatively neutralization-sensitive tier 1A and tier 1B HIV-1 strains; and (ii) several months later, some monkeys also develop NAbs able to suppress the more difficult-to-neutralize tier 2 clade B or clade A and C HIV-1 isolates.

Fig. 4.

Rhesus monkeys generate cross-reactive NAbs during two phases of the SHIV_AD8 infection. (A) The IC₅₀ neutralization titers in plasma samples collected at various times from macaque CL5E against the indicated HIV-1 strains were determined by reciprocal dilution and assay in TZM-bl cells. Values represent reciprocal serum dilution required to achieve 50% neutralization. Value between 20 and 99, green; value between 100 and 999, yellow; value ≥1,000, red. (B) Cross-reactive anti-SHIV_DH12 NAbs rapidly develop in two representative SHIV_AD8–infected animals. Plasma samples, collected from SHIV_AD8–infected macaques DCF1 or CL5E at the indicated times, were diluted 1:20 and assayed for neutralizing activity against pseudovirions carrying the SHIV_DH12-CL-7 (15) envelope glycoprotein in TZM-bl cells.

SHIVs Bearing the HIV-1_AD8 Envelope Glycoprotein Are More Efficient in Generating Cross-Reacting NAbs than a SHIV Expressing the HIV_DH12 env Gene During Infections of Rhesus Macaques.

Two earlier studies have reported cross-reactive anti–HIV-1–neutralizing activity developing in a small fraction of SHIV–inoculated animals during the chronic phase of their infections (19, 20). The availability of preserved plasma samples from large cohorts of the X4-tropic SHIV_DH12–infected monkeys (36 animals) or the R5-tropic SHIV_AD8–infected monkeys (34 animals not evaluated in Fig. 3) provided the opportunity to directly compare the levels of cross-reacting plasma-neutralizing activity generated in macaques inoculated with SHIV_DH12 or SHIV_AD8 against a common heterologous tier 1B clade B HIV-1 isolate (HIV-1_SS1196.1) (21). As shown in Fig. 5, only 1 of 36 SHIV_DH12–infected animals generated IC₅₀ NAb titers >1:100 (a titer of 1:146) against HIV-1_SS1196.1 compared with 29 of 34 SHIV_AD8–infected macaques (titers ranging from 1:105 to 1:1,707) against HIV-1_SS1196.1. In this single head-to-head comparison, the SHIV_AD8 envelope glycoprotein, expressed during in vivo infections, was clearly superior in eliciting cross-reactive anti–HIV-1 NAbs.

Fig. 5.

SHIV_AD8 is far superior to SHIV_DH12 in eliciting potent cross-reactive NAbs during infections of rhesus macaques. Anti–HIV-1_SS1196.1 IC₅₀ neutralizing titers in plasma collected from the indicated infected monkeys were determined by reciprocal dilution and assay in TZM-bl cells. Values represent reciprocal serum dilution required to achieve 50% neutralization. Value between 20 and 99, green; value between 100 and 999, yellow; value ≥1,000, red.

Discussion

The most unique and unexpected property of the SHIV_AD8 family of viruses revealed in this study was its capacity to induce cross-reactive anti–HIV-1 NAbs in a majority of infected animals. Monkey CL5E, which produced NAbs against clade A, B, and C HIV-1 strains, and macaques DCF1 and DA55, which generated IC₅₀-neutralizing titers >1:100 against the tier 2 HIV-1_JR-FL isolate, represent three SHIV_AD8–infected animals that developed cross-reacting NAb responses of significant breadth. Unlike the development of NAbs in many HIV-1–infected persons (22–24), macaques infected with the SHIV_AD8 family of viruses generated cross-reactive NAbs well before or in lieu of autologous NAbs.

There have been at least two earlier studies reporting that some SHIV–infected macaques develop cross-reactive neutralizing activity. One showed that the X4-tropic SHIV_HXB2 or SHIV_89.6 and its derivatives generated NAbs against the exquisitely sensitive T-cell–adapted HIV-1_MN and HIV-1_SF162 strains (20). The cross-reactive neutralizing activity produced by SHIV_HXB2–infected monkeys became measurable 76–124 wk PI but not after 21–41 wk of infection. The second study, monitoring anti–HIV-1 NAbs developing in animals infected with the R5-tropic SHIV_SF162-P4, reported that autologous neutralization activity appeared within the first month of infection in 11 of 12 macaques and that cross-reactive NAbs became detectable in only 3 of 12 monkeys between 6 and 10 mo PI, with the highest titers directed against the tier 1 HIV-1_MN and HIV-1_NL4-3 strains (19).

The temporal appearance of cross-reacting neutralizing activity in SHIV_AD8–infected macaque CL5E was quite intriguing. At face value, at least two bursts of NAb production occurred in this animal, each apparently targeting HIV-1 strains with different neutralization sensitivities. The first was directed against the sensitive tier 1B HIV-1_SS1196.1 (Fig. 4A) and occurred between weeks 5 and 13 PI. Two SHIV_AD8–infected macaques also generated cross-reactive NAbs against the neutralization-sensitive SHIV_DH12 in the same time frame (between weeks 5 and 10 PI) (Fig. 4B). In contrast, the second wave of NAb production in animal CL5E was delayed until week 41 PI, reaching a peak at week 51, and was directed against more neutralization-resistant HIV-1 isolates. The nearly identical emergence times of the second burst of cross-reactive NAbs suggest that the same conserved epitope may be targeted in each of these viruses. It should be pointed out that the IC₅₀ neutralization titers generated against both the sensitive or the more resistant HIV-1 strains generally correlated with levels of set-point viremia in the SHIV_AD8–infected monkeys (Fig. 1 and refs. 13 and 14). For example, macaques DC6W, BIH, CK5T, and DC4N, with some of the highest neutralization titers against the tier 1B SS1196.1 HIV-1 isolate (Figs. 3 and 5), all had set-point plasma viral loads between 10³ and 10⁴ viral RNA copies/mL, whereas animal JLD, with a lower level (∼200 RNA copies/mL) of plasma viremia (Fig. 5), had a very low neutralization titer against the SS1196.1 strain. All three macaques (CL5E, DCF1, and DA55) producing NAbs against the more resistant tier 2 HIV-1 isolates had set-point plasma viremia levels between 10³ and 10⁴ RNA copies/mL.

The delayed appearance of phase 2 neutralization activity in macaque CL5E is also reminiscent of the emergence of the previously described potent cross-clade NAbs generated by SHIV_AD8–infected monkey CE8J, which first became detectable between weeks 32 and 36 PI (12). It is worth noting that a similar two-phase pattern of cross-reacting NAb development has been reported in a longitudinal study of HIV-1–infected individuals (25). The emergence of the second phase required ∼2.5 y during these HIV-1 infections whereas SHIV_AD8–infected animals developed activity against more difficult-to-neutralize HIV-1 strains within 1 y of virus inoculation.

Although a time course of NAb development was not determined for each of the 11 SHIV_AD8–infected animals listed in Fig. 3, it is quite likely that, in addition to monkey CL5E, macaques DCF1 and DA55, both of which generated neutralization IC₅₀ titers >1:100 against the relatively resistant tier 2 HIV-1_JR-FL isolate, also produced both early and late-phase NAbs. Thus, of the 11 SHIV_AD8–infected animals intensively evaluated for the development of cross-reacting NAbs (Fig. 3), 3 monkeys likely exhibited a “two-phase” phenotype of NAb development, and 8 monkeys were able to neutralize only sensitive HIV-1 strains. It is not currently known whether or not a common epitope is targeted by the early emerging cross-reactive NAbs in the latter group of SHIV_AD8–infected animals.

At present, we do not know which epitopes associated with the SHIV_AD8 envelope glycoprotein are driving the production of cross-reactive neutralizing activity or which epitopes in heterologous HIV-1 strains are targeted for neutralization. Because a majority of SHIV_AD8–infected monkeys are able to generate cross-reactive neutralizing activity against multiple HIV-1 isolates, studies of B-cell maturation and the generation of broadly reacting NAbs, triggered by a single HIV-1 envelope glycoprotein (namely HIV-1_AD8) are now possible using the macaque model. One could envisage experiments monitoring B-cell maturation patterns and NAb development in the three SHIV_AD8–infected monkeys (CL5E, DCF1, and DA55 in Fig. 3) able to neutralize the tier 2 HIV-1_JR-FL isolate at IC₅₀ titers >1:100. The results obtained from such a study could guide the development of immunogens designed to elicit late-phase cross-reactive anti–HIV-1 NAbs.

The failure of many SHIV_AD8–infected animals to generate potent and sustained autologous NAbs remains an enigma. We recently reported that only 3 of 19 monkeys inoculated with SHIV_AD8 swarm stocks produced autologous neutralizing activity (13), and in the present study only 5 of 8 macaques inoculated with the SHIV_AD8-EO molecular clone developed autologous NAbs (Fig. 2A). Perhaps envelope glycoproteins associated with relatively neutralization-resistant HIV-1 strains like the tier 3 HIV-1_AD8, rather than envelopes from tissue-culture-adapted virus isolates, may present unique epitopes to the immune system, which elicit an ensemble of cross-reactive NAbs that emerge at different times and possess varying potencies.

Materials and Methods

Construction and Characterization of a Pathogenic SHIV_AD8 Molecular Clone.

The strategy used to obtain a potentially pathogenic SHIV_AD8 molecular clone was (i) to amplify env gene-containing segments from a previously described (14) cohort of nine animals, infected with serially passaged SHIV_AD8 derivatives (Table S1); (ii) to construct full-length SHIV molecular clones carrying some of these amplified env genes; and (iii) to assess the infectivity of reconstructed viruses in rhesus PBMC and in inoculated macaques. The detailed construction of SHIV_AD8 molecular clones and the characterization of resulting virus stocks are described in SI Materials and Methods.

Cell Culture.

HEK293T (293T) and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s MEM supplemented with 10% (vol/vol) heat-inactivated FBS. Rhesus monkey PBMCs were prepared and cultured as described previously (26).

Animal Experiments.

Rhesus macaques (Macaca mulatta) were maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals (27) and were housed in a biosafety level 2 NIAID facility. Phlebotomies, euthanasia, and tissue sample collections were performed as previously described (28). BAL fluid lymphocytes were prepared as previously described (29). All animals were negative for the MHC class I Mamu-A*01 allele.

Quantitation of Plasma Viral RNA Levels.

Viral RNA levels in plasma were determined by real-time reverse transcription-PCR (ABI Prism 7900HT sequence detection system; Applied Biosystems) as previously reported (28).

Lymphocyte Immunophenotyping and Data Analysis.

EDTA-treated blood samples and BAL fluid lymphocytes were stained for flow cytometric analysis as described previously (30–32).

Neutralization Assays.

The neutralization activity present in plasma samples collected from rhesus macaques infected with molecularly cloned or swarm SHIV_AD8 stocks were measured in single-cycle infections of TZM-bl cells by Env pseudoviruses as previously described (14, 33–36). In preliminary assays, 1:20 dilutions of plasma samples were incubated with pseudotyped viruses, expressing the SHIV_AD8 CK15-3 amplicon or the env gene derived from SHIV_DH12-Cl7 (15) and prepared by cotransfecting 293T cells with pNLenv1 and pCMV vectors expressing the respective envelope proteins, as previously described (14). Any plasma sample causing a 50% reduction of luciferase activity compared with that obtained with an uninfected plasma sample was considered positive for NAbs. All experiments were performed in quadruplicate and repeated twice.

The 50% neutralization inhibitory dose (IC₅₀) titer was calculated as the plasma dilution causing a 50% reduction in relative luminescence units (RLU) compared with levels in virus control wells after subtraction of cell control RLU as previously described (18, 33). The neutralization phenotype (tier levels) of the SHIV_AD8 molecular clone was determined by TZM-bl cell assay (18, 33, 36) using plasma samples from a cohort study, which exhibit a wide range of neutralizing activities against subtype B HIV-1 isolates (37).

Neutralization Phenotypes of HIV-1 and SHIV Preparations.

Using standardized plasma pools from HIV-1–infected individuals (18), we determined that the molecularly cloned SHIV_AD8-EO exhibited a tier 2 neutralization phenotype (Table S1), the level usually associated with circulating HIV-1 strains. The parental HIV-1_AD8 exhibited a tier 3 phenotype in this assay. SHIV_DH12CL7 displayed a tier 2 neutralization phenotype with this pool of plasmas.