New SHIVs and Improved Design Strategy for Modeling HIV-1 Transmission, Immunopathogenesis, Prevention, and Cure

SHIV infection of Indian rhesus macaques is an important animal model for studying HIV-1 transmission, prevention, immunopathogenesis, and cure. Such research is timely, given recent progress with active and passive immunization and novel approaches to HIV-1 cure.

substituted by bulky aromatic residues like Trp. In testing this hypothesis, we discovered that substitution of a single residue, 375-Ser, in primary or T/F HIV-1 Envs by Trp, Phe, Tyr, His, or Met resulted in SHIVs that exhibited enhanced binding to rhesus CD4, increased infection of primary rhesus CD4 1 T cells in culture, and consistent infection and replication by SHIVs in RMs in vivo (35). Importantly, these amino acid substitutions at residue 375 did not alter the tier 2 neutralization phenotype of the primary Envs, nor did they appreciably alter their sensitivity to bNAbs that targeted any of the canonical bNAb recognition sites, including CD4bs, V2 apex, V3 high mannose patch, or membrane proximal external region (35). Thus, it became possible, for the first time, to prospectively design SHIVs that expressed particular primary or T/F Envs, including those that elicited bNAbs in HIV-1-infected humans, and to explore parallels in the immune responses of rhesus monkeys and humans to essentially identical Env immunogens (38). This EnvD375 design strategy also made possible the development of SHIVs to evaluate preclinical efficacy of novel active or passive vaccination regimens against challenge by viruses bearing homologous or heterologous primary Envs (7)(8)(9)(10).
Here, we extend this work by constructing 10 new SHIVs, each containing a strategically selected primary HIV-1 Env, that we then validate for retention of native antigenicity, tier 2 neutralization sensitivity, and efficient replication in human and rhesus CD4 1 T cells in vitro and in RMs in vivo. We next describe the development and characterization of a panel of nine SHIV challenge stocks, each containing a unique tier 2 primary HIV-1 Env and grown large scale in primary rhesus CD4 1 T cells, for distribution as challenge strains for active or passive vaccine protection trials. We show that these SHIVs can be efficiently transmitted by different mucosal routes (rectal, vaginal, penile, or oral) and that current vaccination regimens and passively administered bNAbs can prevent transmission of these viruses at neutralization titers similar to those reported in the recently concluded human antibody-mediated prevention (AMP) trials (11). Finally, we describe a new second-generation design strategy that simplifies SHIV construction and eliminates extraneous tat1 and env sequences, thereby making the rhesus-SHIV infection model a more readily accessible and useful research tool.
The design strategy for constructing SHIVs is illustrated in Fig. 1A. This construction scheme allowed for the complete extracellular gp140 region of Env plus the transmembrane segment and 9 amino acids (aa) of the cytoplasmic tail (nucleotides 1 to 2153; HXB2 numbering) to be PCR amplified en bloc and subcloned into a chimeric T/F SIVmac766-HIV-1 proviral backbone (35). If sequences were available for vpu in the source material, then the homologous vpu-env gp140 gene segment was amplified and subcloned into the proviral vector, since homologous vpu-env sequences could potentially enhance the efficiency of Env translation. Env375 codon substitutions corresponding to Trp, Phe, Tyr, His, or Met were introduced by site-directed mutagenesis into each SHIV construct, which was then prepared as a large-scale DNA stock and sequence confirmed. Genome sequences for all SHIVs were contributed to GenBank (Table 1). For each of the 10 primary HIV-1 Envs, six variants containing the different Env375 alleles were made, bringing the total number of newly constructed SHIVs to 60. In the course of SHIV constructions, we noted that certain aspects of the design scheme were inefficient, especially the requirement for multiple PCR amplifications and ligations (see Materials and Methods). We also found in SHIV-infected RMs that redundant tat1 and env gp41 sequences of 68 and 21 bp in length, respectively, that were generated as a consequence of the original cloning strategy underwent spontaneous deletion ( Fig. 1A and Fig. 2) (35,38). Thus, we modified the SIVmac766 backbone vector and amplification primers to simplify the PCR amplification step and eliminate the redundant sequences (Fig. 1B). We used this new design strategy to reclone SHIV. CH505 in order to perform a head-to-head comparison of viruses expressed from this new vector compared with the original SHIV design, and to clone a new SHIV containing the HIV-1 CH0694 Env. Plasmid DNA for all SHIVs was transfected into 293T cells and virus-containing supernatants were characterized for p27Ag content and infectivity on TZM-bl cells. For all SHIVs, p27Ag concentrations ranged from 200 to 2,000 ng/ ml. One nanogram of p27Ag is equivalent to approximately 10 7 virions, so SHIV titers were estimated to range from 2 Â 10 9 to 2 Â 10 10 virions per ml. We confirmed these titers by quantifying vRNA and assuming two vRNA molecules per virion. Infectivity titers on TZM-bl cells ranged from 2 Â 10 5 to 2 Â 10 6 per ml, corresponding to an IU to particle ratio of approximately 10 24 . This ratio is typical for 293T-derived HIV-1 and SIV virions (35), and 100-fold lower than for virus stocks propagated in primary rhesus CD4 1 T cells, where between 1 in 100 and 1 in 50 virions are typically infectious on TZM-bl cells ( Table 2) (35).  (73). c T250-4 is one of several env molecular clones from the isolate CRF_AG_250. One of these clones was designated by Ellenberger and colleagues as "250" and contributed to the NIH HIV Reagent Program (catalog number 11594) and to GenBank (accession number EU513189). This "250" env clone number EU513189, when expressed in 293T cells and used to pseudotype an env-minus HIV-1 proviral backbone, yields noninfectious virions in the TZM-bl assay (G.M.S., unpublished). The T250-4 env clone (GenBank accession number MW507842) differs from number EU513189 by six nucleotides and three amino acids and yields highly infectious HIV-1 Env-pseudotyped virions and highly infectious SHIV.T250-4 virions (see Table 2). d SHIV.BG505 exists in two versions with and without an asparagine and potential N-linked glycan at Env residue 332 (35). (Continued on next page) For SHIVs bearing the 10 new HIV-1 Envs, we evaluated the replication efficiency of each of them containing six different Env375 residues in primary activated human and rhesus CD4 1 T cells in vitro (Fig. 3). With the exception of SHIV.AE.40100, which naturally contains the positively charged, aromatic residue Env375-His, none of the SHIVs containing wild-type Ser or Thr residues at position Env375 replicated appreciably in rhesus CD4 1 T cells (Fig. 3). Conversely, all 10 SHIVs with wild-type Env375 residues replicated efficiently in primary activated human CD4 1 T cells. The latter resultefficient replication of SHIVs containing wild-type 375 alleles in human CD4 1 T cellswas an expected finding but was nonetheless critical to demonstrate, since it confirmed that the chimeric SHIVs that we made were capable of supporting replication. We next asked if substitution of the wild-type Env375 allele by one or more aliphatic or aromatic residues (Met, Trp, Phe, Tyr, or His) would support SHIV replication in rhesus CD4 1 T cells. The answer was affirmative for SHIVs expressing each of the 10 HIV-1 Envs (Fig. 3). The differences in virus replication in rhesus CD4 1 T cells between SHIVs expressing wild-type Env375 residues and those expressing bulky aromatic residues was generally quite large, oftentimes resulting in .100-fold differences in p27Ag concentration in culture supernatants at multiple time points throughout the infection (Fig. 3). Among the six different Env375 alleles that were tested, Env375-Trp most consistently supported SHIV replication in rhesus CD4 1 T cells; it was effective in all 10 HIV-1 Env backbones. Env375-Tyr was the second most favored residue, followed by Env375-His or -Phe. It is notable that Trp is also the most conserved Env375 allele across the broad evolutionary spectrum of primate lentiviruses excluding humans and great apes (30). These results thus corroborate and extend a substantial body of scientific literature indicating that SHIVs bearing primary (nonadapted) wild-type HIV-1 Envs rarely replicate efficiently in rhesus cells (1-3, 27-29, 35, 38, 65, 66) and that this restriction can be lifted by substituting a single amino acid at position Env375. In our combined studies (this study plus references 35 and 38), we replaced wild-type Env375 residues in 16 primary HIV-1 Envs-15 of which could not support SHIV replication in RMs-and found in all instances that this substitution alone led to efficient SHIV replication in rhesus animals.
To extend these findings to in vivo analyses, we inoculated 41 RMs intravenously in groups of 3 to 6 animals each, with SHIVs containing one of the 10 selected HIV-1 Envs and an equal mixture of the six Env375 alleles based on p27Ag content (Table 3 and Fig. 4). We used this experimental design for two reasons. First, because target cell availability is not limited in the initial 2 weeks of infection, during which time virus titers increase exponentially (67-70), we could use deep sequencing of plasma vRNA/cDNA to directly compare the relative replication rates of the six Env375 allelic variants in an in vivo competitive setting. Second, it would be impractical and prohibitively expensive to test 60 SHIVs individually in 60 different monkeys and, even if this could be done, the results would be confounded by monkey-specific variables such as MHC class I and II recognition. Each of the 41 RMs that we inoculated with an SHIV Env375 mixture became productively infected after a single challenge (Fig. 4). In most animals, peak viremia occurred at day 14 post-SHIV inoculation and plasma virus load setpoints were reached 16 to 24 weeks later. Animals treated with anti-CD8 monoclonal antibody (Mab) at the time of SHIV inoculation developed significantly higher peak and setpoint viremia titers compared with untreated animals (P , 0.01 for both). A subset of animals was treated with anti-CD8 MAb at setpoint, 20 to 50 weeks after infection, and most of these animals exhibited increases in virus titers. We performed next-generation sequencing (NGS) on plasma samples taken 2 and 4 weeks postinfection to determine the relative replication rates of the different Env375 allelic var-

FIG 1 Legend (Continued)
A vpu-env amplicon is subcloned into this plasmid vector as described (35). Note in the expanded figures in panel A that the proviral backbone design results in duplications of SIV and HIV-1 tat1 and gp41 sequences (indicated by slash marks) that are spontaneously deleted during in vivo replication of the SHIVs (Fig. 2). The second-generation design scheme (panel B) eliminates these duplications and adds a BsmBI cloning site that allows for simple introduction of either a vpu-env amplicon or an env amplicon. iants (Fig. 4). We expected that differences in infectivity of the Env375 variants would be reflected in the plasma virus quasispecies by 2 weeks postinoculation, since the combined half-lives of circulating virus and the cells producing it is ,1 day (71), resulting in multiple rounds of de novo virus infection and replication during this early interval. This was indeed the case. Overall, there was a good correlation between Env375 residues that supported SHIV replication in vitro and in vivo. For example, in all 10 different Env backgrounds, Env375-Ser failed to support SHIV replication in primary rhesus CD4 1 T cells in vitro (Fig. 3) and the same was true in RMs in vivo (Fig. 4). Conversely, Env375-Trp supported SHIV replication in all 10 Env backgrounds in vitro and was a predominant allele supporting efficient SHIV replication in 7 of 10 Env backgrounds in vivo. There were some differences in Env375 residues that best supported SHIV replication in vitro versus in vivo. For example, for SHIVs bearing ZM233 and CH0694 Envs, 375-Trp supported efficient virus replication in vitro but not in vivo, where 375-Tyr was dominant. And the Env375-His allele, which is naturally present in most subtype AE viruses, including the AE.40100 strain, supported efficient SHIV.AE.40100 replication in rhesus CD4 1 T cells in vitro but not in vivo. Taken together, the findings indicate that substitution of wild-type Env375 alleles in primary HIV-1 Envs with Trp, Tyr, or His results in SHIV chimeras   that replicate efficiently in RMs. However, since it is impossible to predict with certainty which Env375 allele will best support in vivo replication of an SHIV bearing any particular HIV-1 Env, an in vivo competition experiment similar to that illustrated in Fig. 4 must be conducted.
We also compared the relative replication efficiency of SHIV.CH505.375H generated by the firstand second-generation construction strategies (Fig. 5). We showed previously that, in animals infected by viruses produced from the first-generation design, redundant tat1 and env gp41 sequences (68 and 21 bp, respectively) were spontaneously deleted following prolonged in vivo replication ( Fig. 2) (35,38). This suggested a fitness disadvantage for viruses containing the redundant sequences, leading us to hypothesize that animals infected by an equal mixture of the viruses derived from the two designs would show preferential replication by viruses lacking the redundant sequences. This was indeed the case ( Fig. 5A and B). At 3 weeks postinfection, viruses lacking the redundant sequences comprised .95% of the plasma virus quasispecies, and by week 8, they comprised .99% of plasma virus.
To be a relevant model for HIV-1 vaccine studies, SHIV Envs should exhibit clinically relevant antigenic profiles, neutralization sensitivity phenotypes, and coreceptor usage indistinguishable from the primary HIV-1 Envs from which they were derived. We evaluated the neutralization sensitivity patterns of Envs expressing the wild-type Env375 allele compared with Envs expressing one or more of the alternative Env375 alleles that were found to support replication in rhesus CD4 1 T cells in vitro (Fig. 3) and in RMs in vivo (Fig. 4). SHIVs were analyzed using polyclonal anti-HIV-1 sera and a battery of monoclonal antibodies (MAbs) that bind canonical bNAb epitopes, linear V3 epitopes, or CD4induced (CD4i) epitopes (Fig. 6). Linear V3 and CD4i epitopes are generally concealed on native Env trimers from primary viruses (40,(72)(73)(74), and thus MAbs targeting these epitopes typically fail to neutralize primary virus strains. Conversely, neutralization by linear V3 or CD4i MAbs is generally an indication of a nonnative "open" trimer structure, typical of laboratory-adapted viruses. In none of the 10 primary Env backbones that we tested did Env375 substitutions result in neutralization by linear V3 or CD4i MAbs (Fig. 6). Nor did Env375 mutations alter the neutralization sensitivity of these Envs to HIVIG B, HIVIG C, or a high titer, broadly neutralizing HIV-1-infected patient plasma specimen CH1754.   These results suggest that the Envs bearing residue 375 substitutions retained their native or near-native conformation. These Envs also retained their antigenicity with respect to bNAb epitope presentation, since MAbs targeting CD4bs, V2 apex, V3 high mannose patch, and MPER sites exhibited similar neutralization patterns against wildtype and Env375 substituted variants. It is notable that the contours of the neutralization curves, the 50% inhibitory concentration (IC 50 ), IC 80 , and IC 90 values, and the steep sigmoidal inflections, were generally indistinguishable between wild-type Envs and Envs bearing residue 375 substitutions. SHIV.Q23.17 demonstrated neutralization sensitivity patterns to the bNAb MAbs, the three polyclonal anti-HIV IgG and plasma reagents, and the MAbs targeting linear V3 or CD4i epitopes that were similar to the other nine SHIVs, thus supporting a tier 2 status for this virus. We also tested SHIV.CH505.375H derived by firstand second-generation design schemes for sensitivity to HIV-1 bNAbs, the CD4i MAb 17b, HIVIG-C, and the anti-HIV-1 broadly neutralizing polyclonal plasma CH1754; the two virus preparations showed indistinguishable neutralization sensitivity patterns (Fig. 5C). Finally, the SHIVs containing the 10 new HIV-1 Envs were tested for coreceptor usage by analyzing their sensitivity to AMD-3100 (a CXCR4 inhibitor) and Maraviroc (a CCR5 inhibitor). Maraviroc, but not AMD-3100, inhibited the entry of all 10 SHIVs in the TZM-bl entry assay ( Fig. 7), thus demonstrating CCR5-dependent entry. Altogether, the results indicate that Env375 substitutions did not appreciably alter the antigenicity, tier 2 neutralization sensitivity, or CCR5 tropism of any of the 10 SHIVs. SHIVs intended for use as challenge strains in preclinical vaccine trials can be generated from 293T cells by transfection of proviral DNA or by virus passage and expansion in primary human or rhesus CD4 1 T cells. Each approach has potential advantages and disadvantages (3,75). We chose to prepare challenge stocks by infecting primary, activated rhesus CD4 1 T cells with molecularly cloned virus derived from 293T cell transfections and then expanding the virus as rapidly as possible, so as to minimize chances for culture adaptation. By this means, we could ensure that the viral envelopes of challenge stocks contained exclusively rhesus (not human) membrane-associated proteins and that glycosylation patterns would be of rhesus (not human) origin. We selected nine SHIV strains for large scale expansion in rhesus cells and these are listed in Table  2. These SHIVs were chosen to be representative of global HIV-1 diversity, including subtypes A, B, C, D, and AG, and to include SHIVs bearing BG505.N332, CH505, and 1086 Envs, which correspond to vaccine candidates in current or recent human clinical trials. Our aim was to generate large numbers of identical replicates of each SHIV stock (.1,000 vials per SHIV), which could then be characterized biophysically for genetic composition, particle content, infectivity, antigenicity, and neutralization sensitivity and cryopreserved in vapor phase liquid nitrogen (,160°C) for subsequent distribution to the wider scientific community as validated, standardized SHIV challenge stocks. Thus, we inoculated cultures of 100 to 200 million primary, activated, rhesus CD4 1 cells pooled from three naive Indian RMs at a multiplicity of infection (MOI) of approximately 0.01 with genetically homogeneous, sequence-confirmed, 293T transfectionderived virus stocks. For SHIV.CE1176, we infected primary rhesus cells with an equal mixture of Env375-His, Phe, and Trp alleles, and for SHIV.T250-4 we infected cells with an equal mixture of Env375-His, Tyr, and Trp alleles, because no one of these allelic variants had shown preferential replication in all animals tested (Fig. 4). The other SHIV challenge stocks were generated with single Env375 alleles ( Table 2). On days 7 and 14 post-SHIV inoculation, we added new medium and approximately 100 to 200 million fresh, uninfected rhesus CD4 1 T cells from three different naive RMs so as to expand cell numbers and culture volumes while maintaining cell concentrations between 1 and 2 million per milliliter. Beginning on day ;10 post-SHIV inoculation, we collected the total volume of culture supernatant and replaced it with a greater volume of fresh  (Table 2) and in RMs in vivo (Fig. 4).  medium. This complete medium collection and replacement was then repeated every 4 days through day 21. By this means, we could collect as much as 2.5 liters of culture medium containing each SHIV over a period of approximately 21 days. Each supernatant collection was centrifuged twice at 2,500 rpm for 15 min to remove any residual cells or cell debris and then immediately frozen in bulk at 280°C. Supernatants were not filtered, so as to retain the highest possible infectivity titers. Thus, most of the virus that was collected and frozen during the 18 to 21 day culture period was ,4 days old and underwent only one freeze-thaw cycle prior to final vialing. After all supernatant collections had been made, they were thawed at room temperature, combined in a sterile 3-liter flask to ensure complete mixing, and then aliquoted into as many as 2,500 cryovials, generally at 1 ml per vial. The vials were then transferred to vapor phase liquid nitrogen for long-term storage. By this means, we could ensure that all vials for any particular SHIV challenge stock were virtually identical in their contents. Between 192 and 2,224 vials per SHIV, each containing between 0.25 and 1.0 ml of challenge stock, were cryopreserved ( Table 2). Validation analyses were done on thawed cryovial samples to ensure results would be representative of all cryopreserved samples. Challenge stocks were free of bacterial or fungal contamination based on culture on thioglycolate broth. The p27Ag concentrations ranged from 73 to 634 ng/ml and vRNA concentrations ranged from 5.0 Â 10 8 to 4.1 Â 10 9 vRNA/ml. Infectivity was tested on TZM-bl cells, where it ranged from 1.5 Â 10 5 to 3.2 Â 10 7 IU/ml, and on primary rhesus CD4 1 T cells, where it ranged between 1.9 Â 10 3 to 4.1 Â 10 6 IU/ml. The genetic composition of the SHIV challenge stocks was analyzed by single genome sequencing of 39 half-genomes to validate the authenticity of each stock and to determine if there was evidence of selection in vitro (Fig. 8A). Stocks of SHIV.CE1176 and SHIV.T250-4 were sequenced by Illumina deep sequencing to determine the relative proportion of the different Env375 alleles in the final challenge stocks (Fig. 8B). Envelope sequence mean and maximum diversity averaged 0.05% (range 0.03 to 0.13%) and 0.30% (range 0.15 to 0.42%), respectively, in the nine challenge stocks. Mutations across the complete gp160 were essentially random in all challenge stocks except in a secondary expansion of SHIV.CH505. This challenge stock was prepared by infecting naive rhesus CD4 1 T cells with virus from the first expansion of SHIV.CH505 in an attempt to expand sequence diversity and increase infectivity titers. Maximum sequence diversity and maximum sequence divergence from the T/F sequence were 0.35% and 0.29% for stock number 2 compared with 0.15% and 0.08%, respectively, for stock number 1. The p27Ag and vRNA concentrations and infectivity titers on TZM-bl cells were similar for stocks number 1 and number 2 and infectivity titers on primary rhesus CD4 1 T cells were about 3-fold higher for stock number 2 compared with stock number 1.
HIV-1 strains produced in primary human CD4 1 T cells, compared with the same viruses produced in 293T cells, have been reported to exhibit variably greater resistance to neutralizing antibodies (76,77). These differences have been attributed to differences in Env content, cell adhesion molecules, surface glycan composition, or other factors (75). We tested six SHIVs (BG505, CH505, CH848, B41, D.191859 and 1086) produced in primary rhesus CD4 1 T cells and in 293T cells for sensitivity to 17 neutralizing MAbs that targeted CD4bs, V3 glycan, V2 apex, MPER, surface glycan, CD4i, or linear V3 epitopes (Fig. 9). None of the viruses, regardless of cell derivation, were sensitive to the four MAbs that targeted CD4i or linear V3 epitopes, indicating that they retained a native-like closed Env trimer regardless of the cell of origin. SHIVs produced in 293T cells and primary rhesus cells also exhibited similar overall patterns of sensitivity to the other 15 MAbs, in that if an SHIV was sensitive (or resistant) to neutralization by a par-  (Table 3). ticular MAb, then this was true regardless of its cell of origin. However, as reported for HIV-1 strains, we observed enhanced resistance to some MAbs by some SHIVs grown in primary rhesus cells compared with 293T cells. This difference was 2-to 5-fold for all six SHIV strains when exposed to VRC01 and 3BNC117 and as much as 25-fold for certain other virus-antibody combinations, such as BG505-CH01, CH505-CH01, CH505-PGT145, CH505-VRC26.25, B41-CH01, B41-VRC26.25, 1086-PGT128, and 1086-VRC26.25 (Fig. 9).
The properties of rhesus CD4 1 T cell-grown SHIV challenge stocks as summarized in Table 2, especially their consistently high virus titers and infectivity measurements, suggested that these virus strains might be suitable for mucosal transmission studies and to assess the preclinical efficacy of actively induced or passively administered bNAbs. Nearly all natural routes of HIV-1 acquisition result from transmission across mucosal surfaces, the exceptions being intrauterine and intravenous infections. Previously, we showed that SHIVs BG505, CH505, and D.191859 can be transmitted efficiently across rectal, vaginal, and oral mucosae (17,35), resulting in productive clinical infection with virus replication kinetics and plasma virus titers indistinguishable from human infections by HIV-1 (69,70). Penile acquisition is another important route of HIV-1 transmission in humans (78), and Fig. 10A shows that SHIV.D.191859 can be transmitted by atraumatic inoculation of foreskin and glans. Peak viremia occurred at approximately 2 weeks postchallenge and plasma virus load setpoint was reached by 6 weeks. Setpoint viremia persisted at 50,000 to 200,000 vRNA molecules per milliliter through 16 weeks of follow-up, when the experiment was terminated per protocol. These kinetics of SHIV.D.191859 replication post penile transmission were similar to plasma virus load kinetics of the same SHIV strain transmitted by intrarectal, intravaginal, and intravenous routes (Fig. 10A).
Lastly, we performed a low-dose, repetitive challenge rectal titration of SHIV.BG505. N332 in 12 naive RMs to estimate the 50% animal infectious doses (AID 50 ) of the challenge stock and to assess plasma viral load kinetics following IR infection. Three of four inoculations at a dose of 1:20 (1 ml), 3 of 4 inoculations at a dose of 1:100 (1 ml), and 3 of 9 inoculations at a dose of 1:160 (1 ml) resulted in productive clinical infection. Acute and early SHIV.BG505.N332 replication kinetics (Fig. 10B) were similar to mucosal infection by SHIV.D.191859 (Fig. 10A) and also similar to the 10 SHIVs illustrated in Fig.  4 that were infected intravenously. Although our intrarectal AID 50 titration experiment for SHIV.BG505.N332 involved a small number of animals (n = 12) and was subject to stochastic effects related to intrarectal virus inoculation, we could nonetheless estimate the AID 50 of this stock to be approximately 1:120 (1 ml) for atraumatic IR challenge. This result was corroborated in the control (sham-treated) arm of a preclinical trial assessing the protective efficacy of BG505 SOSIP vaccine-elicited neutralizing antibodies against a homologous SHIV.BG505.N332 challenge (7).

DISCUSSION
In recent years, there have been notable advances in HIV prevention and cure research (79-83), yet the goals of effective vaccination and cure-even a "functional"  D  2  5  -7  4  4  8  E  0  1  8  2  1  T  G  P  1  2  1  T  G  P  PGT145 HIVIG . The x axis depicts antibody concentration (mg/ml) except for CH1754 plasma, which is depicted as a percentage dilution.
New SHIVs for Modeling HIV-1 Prevention and Cure Journal of Virology cure-seem far in the distance. Increasingly, experimental medicine trials in humans have been pursued as a strategy to accelerate translational research (82) but, at the same time, there remain untapped opportunities and needs for animal models to complement and synergize with human studies to hasten progress. Different scientific questions demand different model systems, ranging from transgenic or humanized mice to outbred small and large animals. Aside from the great apes, which are endangered and thus precluded from laboratory investigation, the rhesus macaque monkey (Macaca mulatta) is most similar to humans in its immune repertoire (84,85). For HIVrelated investigations in primates, two classes of viruses are broadly used: (i) simian immunodeficiency viruses (SIV) and (ii) chimeric SHIVs that express HIV-1 Envs within an SIV background (3). The present study (i) adds 10 new SHIVs to the research portfolio of HIV investigators; (ii) characterizes key biological properties of these SHIVs that are relevant to virus transmission, prevention, immunopathogenesis, and cure research; and (iii) describes a new SHIV design strategy and cloning vector that can facilitate future SHIV constructions. The HIV-1 Env glycoprotein is critical to virus transmission, persistence, and pathogenesis since it conveys the essential functions of receptor and coreceptor binding and membrane fusion. At the same time, Env is the target of an array of neutralizing antibodies and cytotoxic T cells that cause it to evolve continuously in order to escape recognition that would otherwise lead to virus elimination (38,86,87). Env accomplishes the latter by means of highly evolved properties, including occlusion of trimerinterface epitopes (88), epitope variation (89), conformational masking (90), and glycan shielding (91). Although HIV-1 Env is notorious for its variability and global diversity (www.hiv.lanl.gov), it is nonetheless constrained in its potential for immediate or nearterm evolution due to the myriad of essential biological functions encoded in its sequence (38,(92)(93)(94). These constraints can be lifted, however, by prolonged in vitro cultivation (66) or extensive passage in unnatural animal hosts (1-3, 22). The implication of these observations is that the most relevant HIV-1 Envs (95) for studies of vaccine-elicited protection, passively acquired antibody protection, or curative intervention are primary or T/F Envs from viruses that are responsible for clinical transmission and the establishment of persistent infection in humans (7)(8)(9)(10)96). T/F Envs express the precise primary, secondary, tertiary, and quaternary protein structures that are essential for transmission and T/F Envs are the ones that a vaccine-elicited bNAb response must recognize if it is to be protective (38, 73, 82). Envs derived from short-   (Table 1), were constructed using T/F Envs. The remainder was constructed using primary Envs. A recent study by Keele and colleagues (29) aimed to create new subtype C T/F SHIVs using 20 South African subtype C T/F Envs and either of two strategies to enhance replication in primary RM CD4 1 T cells. One of these strategies was the same EnvD375 design employed here and the other was an EnvD281 approach reported elsewhere (28). Because the O'Brien et al. study (29) pooled SHIVs for competitive replication analyses in RMs, a precise determination of the proportion of wild-type HIV-1 Envs that could support SHIV replication in monkeys could not be made. However, in the instances where EnvD375 substitutions were made and the resulting SHIVs were tested individually, EnvD375 substitutions were successful in conferring replication competence to SHIVs in rhesus cells. The addition of EnvD281 was neither additive nor synergistic. In our studies described here (Table 1) and elsewhere (35,38), we created a total of 16 EnvD375 SHIVs, and each one replicated efficiently in RM CD4 1 T cells in vitro and in RMs in vivo. Altogether, the results suggest that EnvD375 substitution is an effective means for creating SHIVs that have a high likelihood of replicating efficiently in RMs. The second-generation design strategy illustrated in Fig. 1B and Fig. 2 can facilitate this process by substantially reducing the time and effort required to construct new SHIVs and by improving their replication fitness.
A useful outbred primate model for HIV-1 infection of humans should be rational in design, amenable to iterative changes in the challenge viruses, and consistent in reproducing relevant features of disease. Previously, SHIV infections of RMs did not always meet these requirements since SHIVs replicated variably in RMs and often required in vitro or in vivo adaptations to achieve consistent infection or replication. Oftentimes, these changes were not fully understood mechanistically, nor were their immunobiological effects fully appreciated. Moreover, in vitro measures of virus content, infectivity, and replication in cell culture did not always predict in vivo outcomes, lending a measure of uncertainty to SHIV design and analysis. The EnvD375 strategy alleviates much of this uncertainty and unpredictability as demonstrated by the following results: (i) Env375 substitutions alone were sufficient to enhance Env affinity to rhesus CD4, reduce the energetic threshold for downstream Env transitions following CD4 binding, and convey efficient infectivity to the virus in primary rhesus CD4 1 T cells in vitro and in vivo; (ii) the Env375 substitution strategy has worked consistently in that every attempt that we (Table 1), Keele (29), and Barouch (97) have made to engineer a T/F or primary HIV-1 Env SHIV by residue 375 substitution has succeeded in producing a chimeric virus that replicates efficiently in RMs; (iii) the ability of such EnvD375 SHIVs to replicate in vivo was, in each case, predicted by efficient replication in primary, activated rhesus CD4 1 T cells in vitro and, as this is a different result from what has been reported for other SHIVs (1-3, 65), we suspect that our simple EnvD375 design scheme, our protocol for rhCD41 T cell activation, and our method for infecting these cells in tissue culture are responsible for the differences; (iv) the antigenicity and tier 2 neutralization sensitivity of wild-type HIV-1 Envs was closely mirrored by EnvD375 mutants expressed from 293T cells or as infectious SHIVs from primary rhesus CD4 T cells; (v) the genetic diversity of each SHIV infection stock was very low when virus was  (vii) acute and early SHIV replication dynamics in RMs measured by plasma vRNA replicated what has been seen in humans, including a 7 to 14 day eclipse period before vRNA is detectable in plasma, an exponential increase in plasma virus load to a peak approximately 14 to 28 days postinfection, establishment of setpoint viremia two or more months later, and immunopathogenesis leading to clinically defined AIDS in a subset of animals (69,70,73); (viii) SHIV-infected RMs consistently elicited autologous, strain-specific NAbs, and in some cases bNAbs, with kinetics similar to HIV-1-infected humans (35,38); and (ix) molecular pathways of SHIV Env evolution in RMs closely mirrored evolution of homologous HIV-1 Envs in humans, including precise molecular patterns of Env-Ab coevolution leading to Nab escape and, in some animals, the development of bNAbs (38). The latter results speak to the native-like structure of SHIV Envs and to homologies and orthologies in human and rhesus immunoglobulin gene repertoires (38,85). Altogether, the findings highlight the reproducibility and relevance of the SHIV EnvD375-infected RM as a model system for HIV-1 infection in humans. There are, however, limitations to the SHIV EnvD375-infected RM as a model for cure studies, since persistent virus replication is variable between different SHIVs and even with the same SHIV in different animals (Fig. 4) (35, 38). SHIVs D.191859, BG505.N332, and CH505 have generally shown the most consistent replication across multiple studies in monkeys that were not treated with anti-CD8 MAb; replication of these and other SHIVs was generally enhanced by about 10-fold by the administration of rhesus anti-CD8 MAb at the time of SHIV inoculation (Fig. 4). On the other hand, a mechanistic understanding of the basis of spontaneous control of SHIV replication in some monkeys but not others could conceivably inform studies of functional HIV-1 cure in humans. Efficient mucosal transmission, leading to productive clinical infection with consistent patterns of plasma viremia, is a critical feature of SHIVs intended for use as challenge strains to test for vaccine efficacy and for mechanistic studies of virus transmission. We tested SHIVs BG505.N332, CH505, D.191859, and T250-4 for mucosal transmission and determined the titer of each challenge stock for 50% animal infectious doses (AID 50 ). For these studies, virus challenge stocks were grown in primary rhesus CD4 1 T cells. Challenge stocks were first subjected to thorough analytical measurements of virus concentration, infectivity, genotypic complexity, and phenotype with respect to coreceptor usage and antigenicity ( Table 2, Fig. 7, Fig. 9). Because of the wide scientific interest of BG505.N332 SOSIP as a vaccine candidate, we conducted a low-dose atraumatic intrarectal (IR) titration study of SHIV.BG505.N332 (Fig. 10B) where we estimated the IR AID 50 of this stock to be approximately 1:120 (1 ml). Burton and colleagues (7) corroborated this estimate by showing that 6 of 6 naive RMs inoculated intrarectally with a 1:20 (1 ml) dose of this same challenge stock, and 9 of 12 naive RMs inoculated intrarectally with a 1:75 (1 ml) dose, became productively infected after a single challenge (7). Importantly, these results demonstrated reproducibility in clinical infectivity titers of the identical challenge stock used at different primate centers and in animals obtained from different breeding colonies. Replication dynamics of SHIV. BG505.N332 following the low-dose intrarectal inoculations were quite similar in the two studies ( Fig. 10B and reference 7): a meta-analysis of the results revealed peak viremia geometric mean titers of 2.7 Â 10 6 vRNA/ml at day 14 postchallenge and plasma viral load setpoint geometric mean titers of 9.2 Â 10 3 vRNA/ml by week 12, with 23 of 24 animals remaining viremic. Pulendran and colleagues used this same SHIV.BG505. N332 challenge stock for low-dose intravaginal (IVAG) challenges in a preclinical protection study in RMs (9). In a control arm of 15 sham-vaccinated RMs, they found the AID 50 to be approximately 1:3 (1 ml). Peak plasma viremia (GMT = 1.7 Â 10 6 vRNA/ml) was again at 14 days postinfection and plasma viral load setpoint was reached by week 10, with 14 of 15 animals remaining viremic (GMT = 1.7 Â 10 3 vRNA/ml). The 40-fold difference in AID 50 between IR and IVAG challenge routes is consistent with previous findings with SHIV and SIVs (3,98,99) and is similar to estimates of relative infectivity in humans exposed to receptive anal intercourse versus receptive vaginal intercourse (78). We also titrated SHIV.CH505 challenge stocks for AID 50 in RMs following intrarectal or intravaginal inoculation. In independent studies with a total of 21 RMs, Klatt (reference 100 and unpublished data) and Haynes estimated the AID 50 following IR challenge of naive RMs to be approximately 1:80 (1 ml), while Felber and colleagues (8) found the AID 50 of this stock following IVAG challenge to be approximately 1:2 (1 ml) ( Table 2). These findings again demonstrate reproducibility in AID 50 titers in different primate centers and in monkeys from different breeding colonies, as well as a 30-to 40-fold difference in infectivity between IR versus IVAG challenge routes. Previously, we estimated the AID 50 for SHIV.D.191879 for IVAG inoculation to be approximately 1:3 (1 ml) (101). Here, we could not estimate an AID 50 for penile transmission by the SHIV. D.191879 challenge stock since 2 of 2 animals became infected after a single inoculation (Fig. 10A), but the findings suggest that the AID 50 titers of this stock for penile transmission are likely to be sufficient for it to be used as a challenge stock in preclinical prevention trials once formal titering is completed. Finally, in an ongoing study, Sok, Rakasz, and colleagues have estimated the AID 50 of SHIV.T250-4 to be approximately 1:160 (1 ml inoculum) following atraumatic rectal inoculation (unpublished data). Thus, in multiple studies of mucosal infection by BG505.N332, CH505, D.191859, and T250-4, AID 50 titers and postinfection plasma viral load kinetics were consistent between SHIVs and between studies conducted at different primate facilities and mirrored analytical assessments of the different challenge stocks in vitro (Table 2). These findings suggest that precise measurements of virion content and infectivity of different challenge stocks correlate well with AID 50 titers following mucosal challenge, which is important because it can facilitate AID 50 titrations of new challenge stocks going forward.
Altogether, the findings of this study suggest that the SHIVs listed in Table 1 can be broadly useful as challenge stocks for preclinical studies of vaccine-elicited or passively acquired antibody protection, for assessing novel cure interventions, and for mechanistic studies of virus transmission and pathogenesis. We have contributed the rhCD4 T cell-grown SHIV challenge stocks and the 16 SHIV plasmid DNA stocks to the NIH NIAID HIV Reagent Repository and to the Penn Center for AIDS Research Virology Core Laboratory, which provide investigators with derivative reagents (e.g., barcoded SHIVs for lineage tracing, sequence-verified viral DNA maxipreps, minimally adapted T/F SHIV variants with enhanced in vivo replication dynamics, and titered 293T-derived infectious virus stocks) to meet research needs. One important research application that we anticipate in the future is in comparative efficacy testing of different vaccines against common heterologous tier 2 primary virus challenge stocks, and the same vaccine against a common heterologous virus administered by different mucosal inoculation routes. Such studies promise to inform HIV-1 immunogen design and testing as new vaccine candidates are developed.

MATERIALS AND METHODS
Ethical statement. Indian rhesus macaques were housed and studied at Bioqual, Inc., Rockville, MD or at the Plum Borough animal facility at the University of Pittsburgh, Pittsburgh, PA, according to guidelines and standards of the Association for Assessment and Accreditation of Laboratory Animal Care and the Animal Welfare Act. Experiments were approved by the Bioqual, University of Pittsburgh, Duke University and University of Pennsylvania Institutional Animal Care and Use Committees. All RMs included in this study were socially housed (paired) indoors in stainless steel cages, had 12/12 light cycle, were fed twice daily, and water was provided ad libitum. A variety of recommended environmental enrichment strategies were employed. The animals were observed twice daily and any signs of disease or discomfort were reported to the veterinary staff for evaluation. For sample collections, animals were anesthetized with 10 mg/kg ketamine HCI (Park-Davis, Morris Plains, NJ, USA) or 0.7 mg/kg tiletamine (HCI) and zolazepan (Telazol, Fort Dodge Animal Health, Fort Dodge, IA) injected intramuscularly. At the end of the study, the animals were sacrificed by intravenous administration of barbiturates.
Nonhuman primate care and procedures. Animals were approximately equally divided male and female, aged 3 to 12 years and negative for Mamu-A*01, B*08, and B*17. Animals were screened to be negative for retroviruses, measles, Ebola virus, and T. cruzi. Animals were sedated for blood draws, anti-CD8 MAb infusions, and SHIV inoculations. For estimations of AID 50 of SHIV challenge stocks, animals were inoculated atraumatically by penile, rectal, or vaginal routes. Penile inoculations were performed in anesthetized animals in a recumbent supine position. The foreskin was retracted vertically and laterally and separated from the glans exposing the preputial mucosa and coronal sulcus. An aliquot of 0.25 ml of undiluted SHIV challenge stock was slowly and carefully inoculated into this potential space using a 1-ml syringe and the vertical-lateral foreskin retraction maintained for 20 min. Intrarectal or intravaginal inoculations were performed by inserting a flexible lubricated pediatric feeding tube atraumatically 3 to 5 cm into the rectum or vagina of animals lying in a Trendelenburg position followed by the administration of diluted virus stock in a total volume of 1 ml by slow push. Animals were maintained in the Trendelenburg position for 30 min before being repositioned and returned to their cages to recover from anesthesia. Intravenous SHIV inoculations were performed by placing an intravenous (i.v.) catheter into the femoral vein of anesthetized animals in supine position, administering small volumes of sterile normal saline and confirming venous access by retrograde blood return. SHIVs bearing any of 10 different HIV-1 Envs, each with as many as six Env375 allelic variants in a total volume of 1 ml Dulbecco's modified Eagle medium (DMEM), were administered by slow push. The different Env375 allelic variants were equilibrated based on p27Ag concentration, since the variable we were testing was relative infectivity as reflected in SHIV replication efficiency and plasma vRNA load in vivo. Thus, a typical inoculum consisted of a total of 300 ng SHIV p27Ag, composed of 50 ng p27Ag of each of six SHIV Env375 variants. Following virus inoculation, the i.v. line was flushed with normal saline. A subset of animals then received an intravenous infusion of 25 mg/kg of anti-CD8alpha MAb (MT807R1; NIH Nonhuman Primate Reagent Resource, NHPRR [https://www.nhpreagents.org/]) or anti-CD8beta MAb (CD8beta255R1; NHPRR) MAb over a period of 3 to 5 min. Another subset of animals received anti-CD8 MAb at 18 to 48 weeks postinfection. RMs were phlebotomized weekly, then monthly, and then every other month to collect and cryopreserve blood samples.
Processing and storage of rhesus and human blood specimens. Blood samples from rhesus macaques were collected in sterile 10-ml vacutainers containing ACD-A anticoagulant. Up to 40 ml of ACD-A anticoagulated blood from each RM was combined in a sterile 50-ml polypropylene conical tube, centrifuged at 2,100 rpm (1,000 Â g) for 10 min at 20°C, and the plasma collected in a fresh 50 ml conical tube without disturbing the buffy coat WBC layer and large red cell pellet. The plasma was centrifuged again at 3,000 rpm (;2,000 Â g) for 15 min at 20°C in order to remove all platelets and cells. Plasma was collected and aliquoted into cryovials and stored at 280°C. The red blood cell (RBC)/WBC pellet was resuspended in an equal volume of Hanks balanced salt solution (HBSS) without Ca 11 or Mg 11 and containing 2 mM EDTA and then divided into four 50-ml conical tubes. Additional HBSS-EDTA (2 mM) buffer was added to bring the volume of the RBC/WBC mixture to 30 ml in each tube. The cell suspension was then carefully underlayered with 14 ml 96% Ficoll-Paque and centrifuged at 1,800 rpm (725 Â g) for 20 min at 20°C in a swinging bucket tabletop centrifuge with slow acceleration and braking so as not to disrupt the Ficoll-cell interface. Mononuclear cells at the Ficoll interface were collected and transferred to a new 50-ml centrifuge tube containing HBSS-EDTA (w/o Ca 11 or Mg 11 ) and centrifuged at 1,000 rpm (;200 Â g) for 15 min at 20°C. This pellets peripheral blood mononuclear cells (PBMCs) and leaves most of the platelets in the supernatant. The supernatant was removed and the cell pellet was resuspended in 40 ml HBSS (with Mg 11 /Ca 11 and without EDTA) 1 1% fetal bovine serum (FBS). To remove additional contaminating platelets, the cell suspension was centrifuged again at 1,000 rpm (;200 Â g) for 15 min at 20°C and the supernatant discarded. The cell pellet was tap-resuspended in the residual 0.1 to 0.3 ml of medium and then brought to a volume of 10 ml HBSS (with Mg 11 /Ca 11 ) plus 1% FBS. Cells were counted and viability assessed by trypan blue exclusion. Cells were centrifuged again at 1,200 rpm (300 Â g) for 10 min at 20°C, the supernatant discarded, and the cells resuspended at a concentration of 5 to 10 Â 10 6 cells/ml in CryoStor cell cryopreservation medium (Sigma number C2999), aliquoted into 1-ml cryovials (CryoClear cryovials; Globe Scientific Inc., number 3010), placed in a Corning CoolCell LX cell freezing container, stored overnight at 280°C, and then transferred to vapor phase liquid N 2 for long-term storage. Alternatively, freshly isolated rhesus PBMCs were processed immediately for CD4 1 T cell purification and activation. Human PBMCs from deidentified normal blood samples were isolated by similar procedures from leukopaks obtained from the University of Pennsylvania Comprehensive Cancer Center Human Immunology Core Laboratory and either cryopreserved or used immediately for CD4 1 T cell purification and activation.
SHIV constructions. SHIVs were constructed in one of two chimeric SIV/HIV proviral backbone plasmids. The original backbone (Fig. 1A) was first described by Li et al. (35) and was used in that study to generate SHIV.A.BG505, SHIV.B.YU2, SHIV.C.CH505, SHIV.C.CH848, and SHIV.D.191859. This backbone was subsequently employed by Roark to generate SHIV.C.CAP256SU (38) and by other investigators to generate still other SHIVs, all based on this EnvD375 design strategy (29,97). We designated the firstgeneration plasmids as pCRXTOPO.SHIV.v1.backbone1 and pCRXTOPO.SHIV.v1.backbone2. Version 1 backbones 1 and 2 allow for the cloning of vpu-env (env nucleotides 1 to 2153, HXB2 numbering) or envonly (env nucleotides 1 to 2153, HXB2 numbering) amplicons, respectively. This first-generation plasmid required cumbersome sequential PCR amplifications and ligations in order to generate a complete replication-competent chimeric SHIV provirus. In addition, the first-generation design scheme generated an SIV/HIV-1 tat1 redundancy and an HIV-1 env gp41 redundancy, both of which were spontaneously deleted when SHIVs replicated persistently in vivo (e.g., see Fig. 2A and references 35 and 38). We thus engineered second-generation SHIV cloning vectors designated pCRXTOPO.SHIV.v2.backbone1 and pCRXTOPO.SHIV.v2.backbone2, which allow for cloning of the identical vpu-env and env-only amplicons, respectively. In the first-generation SHIV backbone, unique restriction enzyme recognition sites for BstBI and XhoI are present in the middle of SIV vpx and after the 39 LTR in the vector sequence, respectively. We synthesized two fragments that contain these two enzyme sites and the genes in between. We eliminated the redundant tat1 and env gp41 sequences and replaced the vpu-env and env genes with a linker fragment that carries two BsmBI restriction enzyme sites (Fig. 1). The BsmBI site appended at the N terminus of the linker recognizes the reverse cDNA strand and creates a 39 overhang; the one added at the C terminus recognizes the positive-strand DNA and creates a 59 overhang. This design results in two different sticky ends, which allows unidirectional cloning of the insert into the backbone. BsmBI is a type IIS restriction enzyme that cleaves outside its recognition site and thus the enzyme recognition sequence does not remain after ligating the insert into the backbone (Fig. 1). The two synthesized fragments were then cloned into the original SHIV backbone separately using the BstBI and XhoI sites. The resulting two SHIV backbones (GenBank accession numbers MW476487 and MW476488) were then used for cloning env (nucleotides 1 to 2153, HXB2 numbering) or vpu-env, respectively. The vpu-env gp140 segments of HIV-1 CE1176, CH1012, T250-4, Q23.17, WITO, ZM233, 1086, B41, and 40100 were cloned into the first-generation SHIV backbone using methods described previously   (35). The vpu-env gp140 segments of HIV-1 CH0694 and CH505 were cloned into the second-generation SHIV backbone by appending the BsmBI recognition sequences to the 59 and 39 ends of the amplicon and performing a standard ligation (35). We then used the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) to create allelic variants (M, Y, F, W, or H) of the wild-type Env375-Ser or -Thr codons. Wild-type and mutant plasmids were transformed into MAX Efficiency Stbl2 competent cells (Invitrogen) for maxi-DNA preparations. Each 10-kb viral genome was sequenced in its entirety to authenticate its identity and genome integrity. Infectious SHIV stocks were generated in 293T cells as previously described.
SHIV infection of primary rhesus and human CD4 + T cells. Purified rhesus and human CD4 1 T cells were isolated from PBMCs using magnetic MACS CD4 MicroBeads (Miltenyi Biotec), as previously described (35). They were activated by incubation with anti-biotin MACSiBead particles (Miltenyi Biotec) loaded with biotinylated anti-CD2, -CD28, and -CD3 MAbs, as previously described (35). The replication kinetics of each of the SHIVs and Env375 variants in primary, activated human and rhesus CD4 1 T cells were determined again as previously described (35). Briefly, 293T supernatants containing 300 ng p27Ag of each variant were added to 2 Â 10 6 purified human or rhesus CD4 T cells in complete RPMI growth medium (RPMI 1640 with 15%) heat-inactivated fetal bovine serum (FBS, HyClone), 100 U/ml penicillinstreptomycin (Gibco), 30 U/ml interleukin 2 (IL-2) (aldesleukin, Prometheus Laboratories), and 30 mg/ml DEAE-dextran. For MOI estimation, 300 ng p27Ag is equal to ;3 Â 10 9 virions, ;3 Â 10 5 IU on TZM-bl cells, or ;3 Â 10 4 IU on primary CD4 1 T cells, so the estimated MOI of this titration was estimated to be between 0.01 and 0.05. The cell and virus mixtures were incubated for 2 h under constant rotation at 37°C to facilitate infection, washed three times with RPMI 1640, and resuspended in complete RPMI 1640 medium lacking DEAE-dextran. Cells were plated into 24-well plates at 2 million cells in 1 ml and cultured for 13 days, with sampling of 0.2 ml supernatant and medium replacement every 2 to 3 days. Supernatants were assayed for p27Ag concentration by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Zeptometrix).
SHIV challenge stock generation in primary rhesus CD4 + T cells. A total of 100 to 200 million primary, activated, rhesus CD4 1 cells pooled from three naive RMs at a concentration of 10 7 cells/ml in complete RPMI 1640 medium with 10% fetal calf serum (FCS) and DEAE-dextran (30 mg/ml) were inoculated with 293T cell-derived SHIVs at an MOI of 0.1 to 0.5 in TZM-bl cells and an estimated MOI of 0.01 to 0.05 in primary rhesus CD4 1 T cells. For SHIV.CE1176, we infected primary rhesus cells with an equal mixture of Env375-His, -Phe, and -Trp alleles, and for SHIV.T250-4 we infected cells with an equal mixture of Env375-His, -Tyr, and -Trp alleles, because these alleles in these two Env backgrounds had shown differential replication in different animals (Fig. 3). The other SHIV challenge stocks were generated with viruses containing single rhesus-preferred Env375 alleles ( Table 2). The total volume of the SHIV-cell mixture was typically 10 to 30 ml, depending of the infectivity titers of the 293T virus stock. The SHIV-cell mixture was transferred to a T75 flask, which was fixed to a rotating wheel or rocker so that leakage or spillage was not possible. This apparatus was then placed in a 37°C in a 5% CO 2 incubator for 2 h of continuous mixing. The contents of the T75 flask were then transferred to a sterile 50-ml polypropylene tube and centrifuged at room temperature at 1,200 rpm (;300 Â g) for 10 min. The supernatant was decanted, the cells gently tap resuspended in the residual medium (,0.5 ml), and then resuspended in 50 ml complete RPMI medium with 10% FCS and the wash step repeated twice. The washing steps are important to remove DEAE-dextran, which can be toxic to cells in culture, and to remove unbound virus. Cells were then resuspended at a concentration of 1 to 2 Â 10 6 cells/ml in complete RPMI 1640 medium with 10% FCS, Il-2, and antibiotics in T100 flasks and incubated at 37°C in a 5% CO 2 incubator. On days ;7 and ;14 post-SHIV inoculation, additional fresh medium and approximately 100 to 200 million fresh, uninfected, activated rhesus CD4 1 T cells from three different naive RMs were added to the cultures, which were transferred into T250 flasks to accommodate larger volumes. This expansion of the cultures markedly increased cell numbers and supernatant volumes while maintaining cell concentrations between 2 and 4 million per milliliter. The culture supernatant was sampled on approximately days 1, 4, 7, 10, 14, 17, and 20 for p27Ag concentration, with assays performed weekly. Typically, p27Ag concentrations were ,50 ng/ml on day 7 but rose rapidly to .200 ng/ml by day 10. On day ;10 post-SHIV inoculation, the total volume of culture supernatant was collected, centrifuged twice at 2,500 rpm (1,000 Â g) for 15 min to remove any residual cells or cell debris, and then frozen in bulk at 280°C. The supernatant was replaced with a greater volume of fresh medium as additional uninfected activated rhesus CD4 1 T cells were added and as cells divided, again keeping cell concentrations at 2 to 4 million per milliliter. Between days 10 and 21, p27Ag production was maximal and concentrations in the supernatant rose rapidly to .200 ng/ml every 3 to 4 days after each complete collection of culture supernatant. By this means, we could collect as much as 2.5 liters of culture medium containing each SHIV over a 3-week culture period. Importantly, because complete supernatant collections and fresh media replacements were performed every 3 to 4 days beginning on day ;10 post-SHIV inoculation, most of the virus that was collected and frozen was ,4 days old and underwent only one freeze-thaw cycle prior to final vialing. Once all supernatant collections had been made over the 18-to 21-day culture period, they were thawed at room temperature at the same time, combined in a sterile 3-liter flask to ensure complete mixing, and aliquoted into as many as 2,500 cryovials, generally at 1 ml per vial. The vials were then transferred to a 280°C freezer overnight and then to vapor phase liquid nitrogen for long-term storage. By this means, we could ensure that all vials were essentially identical in their contents.
Virus entry and neutralizing antibody assays. Assays for virus entry and neutralizing antibodies were performed using TZM-bl indicator cells, as previously described (35,91). The NAb assay is essentially identical to that employed by Montefiori, Seaman, and colleagues (102) (https://www.hiv.lanl.gov/ content/nab-reference-strains/html/home.htm), the only difference being that we plate virus and test plasma or MAbs or purified polyclonal IgG onto adherent TZM-bl cells and hold the concentration of human and rhesus plasma/serum constant across all wells at 10%. In addition to this 10% final concentration of plasma/serum, the culture medium consists of Dulbecco's modified Eagle's medium (DMEM) with 40 mg/ml of DEAE-dextran and pen-strep antibiotics. Infections were performed in duplicate. Uninfected cells were used to correct for background luciferase activity. The infectivity of each virus without antibodies was set at 100%. The 50% inhibitory concentration (IC 50 ) is the antibody concentration that reduces by 50% the relative light units (RLU) compared with the no-Ab control wells after correction for background. Nonlinear regression curves were determined and IC 50 values calculated by using variable slope (four parameters) function in Prism software (v8.0). In the virus entry assay used to determine infectivity titers of 293T cell-derived viruses (Table 2), the culture medium consisted of DMEM with 10% FBS, 40 mg/ml DEAE-dextran, and pen-strep antibiotics and cell entry was quantified by betagalactosidase expression after 48 h, as described (35).
Coreceptor use analysis. TZM-bl cells were seeded in 96-well plates at a density of 15,000 cells per well and cultured overnight at 37°C with humidified air and 5% CO 2 . Cells were incubated with selective entry inhibitors for 1 h, followed by inoculation of 2,000 TZM-bl IU of virus per well. Coreceptor inhibitors included 10 mM Maraviroc (CCR5), 1.2 mM AMD3100 (CXCR4), a mixture of inhibitors, or medium-only control. Viral Envs YU2 (CCR5-tropic) and SG3 (CXCR4-tropic) were included as controls. The infectivity of the medium-only control wells was set at 100%. The infectivity of the experimental wells was quantified by percentage of infection compared with the medium-only control wells after correction for background.
Plasma viral RNA quantification. Plasma viral load measurements were performed by the AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory, and by the NIH/ NIAID-sponsored Nonhuman Primate Virology Core Laboratory at the Duke Human Vaccine Institute, as previously described (35,38). Over the course of this study, the sensitivity limits for accurate vRNA quantification using 0.5 ml of NHP plasma improved from 250 RNA cp/ml to ,100 RNA cp/ml. We chose a conservative threshold of 100 RNA cp/ml for a limit of detection and 250 RNA cp/ml for the limit of quantification.
Viral RNA sequencing. Single genome sequencing of SHIV 39 half genomes was performed as previously described (35,73). Geneious software was used for alignments and sequence analysis. The sequences were visualized using the LANL Highlighter plot tools (https://www.hiv.lanl.gov/content/sequence/ HIGHLIGHT/highlighter_top.html). To analyze the prevalence of 375 variants, next-generation sequencing was performed using the Illumina MiSeq system as described (35,38). For each animal, 20,000 to