Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism.
Product# HIV-1 M/T Tropic Envelope
Product# 1081 HIV-1 gp120 (ADA)
Product# 1011 HIV-1 gp120 (subtype C)
Product# 1031 HIV-1 gp120 (YU2)
The phase III RV144 human immunodeficiency virus (HIV) vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing the HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clade A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: the simultaneous depletion of B and CD8+ cells followed by the intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High-level viremia and CD4+ T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4+ T-cell loss; the partially adapted SHIV had become dual tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5 tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and was pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans.
IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in nonhuman primate (NHP) models requires test viruses that allow the evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to nonneutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive-immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed a coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.
Human immunodeficiency virus (HIV) continues to be a major global public health issue, and attempts to produce a safe and effective vaccine have not yet been successful. Although major research efforts have focused on AIDS vaccine development, a safe and effective vaccine is not yet available. The RV144 vaccine trial resulted in 31.2% protection, making it the first phase III study to show limited but significant efficacy in preventing HIV acquisition (1). Infection-risk analyses revealed that nonneutralizing antibodies (Abs) targeting the HIV Env V1/V2 region correlated inversely with infection risk (2, 3), and subsequent studies identified other nonneutralizing functional activities that also appear to be inversely correlated with risk (4, 5). A number of monoclonal antibodies (mAbs) have been isolated from RV144 vaccine recipients, including mAbs directed against the Env regions linked to lower risks of HIV acquisition (6). However, the potential of these mAbs to prevent acquisition in a relevant nonhuman primate (NHP) model has not yet been assessed.
A number of NHP studies have sought to replicate key findings of RV144 with corresponding simian immunodeficiency virus (SIV) immunogens. Of note, these challenge studies confirmed some of the key findings of RV144 (7, 8). However, anti-HIV Env-directed Ab responses cannot be evaluated in SIV models. A simian-human immunodeficiency virus (SHIV) displaying the HIV envelope circulating in Thailand will represent an important tool to assess active- and passive-immunization strategies in NHP models.
Almost all newly acquired HIV infections involve R5-tropic strains; approximately 90% of new HIV infections occur through mucosal exposures. Most recently transmitted HIV strains are relatively difficult to neutralize and exhibit a tier 2 neutralization phenotype. Consequently, NHP models to evaluate strategies to prevent HIV acquisition should consider these key biological characteristics of HIV acquisition among humans.
SHIVs have been used as tools to evaluate anti-HIV Env Ab responses. Most SHIVs used to date have been built from the backbone of SIVmac239, with the exception of a recently reported strain (9). In general, SHIVs carry HIV vpu, tat, rev, and env. Previously, we generated a panel of non-clade B SHIVs, including several encoding HIV clade C envelopes. Furthermore, we engineered the long terminal repeat (LTR) of SIVmac239 to resemble that of HIV more closely (10). The HIV LTR generally contains at least two, but up to four, NF-κB sites, in contrast to the SIVmac239 LTR, which has only one such site (11). A larger number of NF-κB sites renders proviruses more responsive to stimulation by cytokines that act through the NF-κB pathway, including tumor necrosis factor alpha (TNF-α). In Thailand, where the RV144 trial was conducted, CRF01_AE strains circulate, and Env is predominantly derived from HIV clade E (1, 12).
Here we report the construction, novel in vivo adaptation, and pathogenicity of a SHIV encoding the env gene isolated from a placebo recipient of the RV144 vaccine efficacy trial in Thailand. This SHIV, termed SHIV-E1p5, is R5 tropic, has a tier 2 neutralization phenotype, is mucosally transmissible, and is pathogenic, as indicated by its ability to induce AIDS in NHPs. During adaptation, SHIV-E1 and progeny strains mimicked an important aspect of HIV CRF01_AE, namely, the ability to switch coreceptor usage and become dual tropic or solely X4 tropic. Deep-sequencing analysis of the various virus isolates during adaptation revealed env mutations uniquely associated with dual-tropic or X4-only phenotypes; such mutations were absent in the final R5-only SHIV-E1p5 isolate. Our newly created SHIV-E1 reflects key biological aspects of HIV clade E in humans, and the final isolate, SHIV-E1p5, can be used as a model to develop prevention strategies targeted against CRF01_AE.
Construction of SHIV carrying CRF01_AE env.
To determine the ability of RV144 vaccine-induced Abs to protect against HIV acquisition by passive immunization in an NHP model, we generated a SHIV carrying HIV CRF01_AE env. Six HIV CRF01_AE env clones of recently transmitted viruses isolated from placebo group RV144 participants were tested for infectivity as pseudotyped viruses generated by the cotransfection of HIV CRF01_AE env genes with an env-deleted provirus into 293T cells. All infectious env genes were used to generate SHIV clones according to the construction schema (Fig. 1). Overall, 30 infectious SHIV clones were obtained, as evidenced by the transfection of 293T cells and analysis of cell-free supernatants in TZM-bl cells (data not shown). One of them, SHIV harboring env clone 620345.2, was chosen for further development and renamed SHIV-E1 for the sake of simplicity. The backbone, SHIV-1157ipd3N4 (10), was chosen because it contains a 3′ engineered LTR with a duplication of the NF-κB site. As such, the engineered LTR resembles that of HIV more than that of SIVmac239, which contains only one NF-κB site. Of note, all HIV LTR elements contain at least two NF-κB sites, with different clades containing up to four such sites. The resulting SHIV-E1 was tested by DNA sequence analysis, coreceptor usage, and neutralization phenotype. SHIV-E1 was exclusively R5 tropic and relatively difficult to neutralize, corresponding to a tier 2 neutralization phenotype. Cell-free SHIV-E1, prepared by transfection of 293T cells, replicated in TZM-bl cells, U87.CD4.CCR5 cells, and human peripheral blood mononuclear cells (PBMC) depleted of CD8+ cells. PBMC from rhesus macaques (RMs) (25 donors) and pig-tailed macaques (5 donors) did not support the replication of the parental virus, even after the depletion of CD8+ cells. Clearly, a special approach needed to be designed to achieve successful adaptation to RMs.
Construction of SHIV-E1. (A) Several infectious HIV-E1 env clones were identified from newly infected individuals of the RV144 placebo group. env sequence divergence is shown. (B) To generate the initial parental SHIV-E1 provirus, env clone 620345.2 (0.0042) (red box) was inserted into the SHIV-1157ipd3N4 backbone (10). The latter infectious molecular clone had been engineered to contain duplicated NF-κB sites (NN) in the long terminal repeats (LTRs). As such, this SHIV LTR resembles the HIV LTR better than the original SIVmac239 LTR that contains only one NF-κB site. For the sake of simplicity, we use the term “SHIV-E1” for our initial infectious molecular SHIV clone. Of note, in the RV144 CRF01_AE strains used, the env sequences per se originated from HIV clade E strains rather than clade A strains. Thus, the env clone 620345.2 (0.0042) (shown in red boxes in panels A and B) used to generate the SHIV-E1 construct was renamed “HIV-E1 env.” The cloning sites KpnI (K) and BamHI (B) are shown. TM, transmembrane region.
In vivo transfection and double-immune depletion to adapt SHIV-E1.
To achieve replication of SHIV-E1 in RMs, we did not rely solely on the inoculation of cell-free virus. Instead, we opted for intramuscular (i.m.) inoculation of plasmid DNA encoding infectious SHIV-E1 (Fig. 2A and andBB and and3,3, top left). The rationale for this approach was to generate virus particles displaying RM host proteins on their surfaces after budding from transfected muscle cells. In addition, we inoculated the first animal, animal R547, intravenously (i.v.) with three doses of cell-free SHIV-E1 on alternate days (Fig. 2A and andBB and and3,3, top left). To give the virus the best chance to replicate, we eliminated adaptive immunity by depleting CD8+ cells with a cytotoxic mAb and by depleting B cells with the anti-CD20 mAb rituximab (Rituxan; Genentech Inc.) (Fig. 2A and and3,3, top left). Of note, the anti-CD8 mAb affects both CD8+ T cells as well as the majority of natural killer (NK) cells in RMs, thereby also significantly interfering with host innate immune responses. This approach was successful: peak viremia reached >109 viral RNA (vRNA) copies in the first animal (Fig. 3, top left).
As expected, the depletion of B cells and CD8+ cells resulted in profound lymphopenia, to which the host responded initially with the expected compensatory rise in absolute CD4+ T-cell numbers (Fig 3., top left), reaching a peak of >3,000 cells/mm3 concomitant with a high peak vRNA level, thus providing abundant target cells for SHIV-E1. The latter replicated to very high levels, with the simultaneous destruction of target cells, as evidenced by the precipitous drop of absolute CD4+ T-cell numbers to <200 cells/mm3 on day 53, a clear indication of viral pathogenicity. In vivo transfection with infectious proviral DNA combined with the repeated administration of cell-free virus and double-immune depletion of the host was successful.
Passage of SHIV-E1-infected blood to the second donor and virus isolation.
On day 53 postinoculation, blood from animal R547 was transferred i.v. to the second recipient, animal R551. On day 56 postinoculation, this RM developed neck and facial swelling, which prompted an emergency diagnostic workup. Peripheral blood smears showed severe parasitemia. The animal was euthanized immediately on day 57 (Fig. 3, top right); the parasite was identified subsequently as Trypanosoma cruzi. Animal R551's profound peripheral blood CD4+ T cell loss toward the end of its course may have facilitated severe parasitemia by a known opportunistic pathogen of humans with end-stage HIV disease (13).
In order to continue virus passage, plasma collected at the time of necropsy was filtered to remove T. cruzi and given i.v. to the next two recipients, animals R555 and R561 (passage 3a [P3a] and P3b) (Fig. 2A and andBB and and3,3, bottom). Of note, these two animals were not immunodepleted because the virus in the previous donor, R551, had replicated robustly. At the time of necropsy, virus was also isolated from animal R551 by cocultivation of PBMC, resulting in SHIV-E1p2d57. We conclude that after the first two passages, virus adaptation had succeeded, resulting in a highly replication-competent virus that eliminated CD4+ T cells in the first two doubly immunodepleted recipients.
Unexpected rapid decline of CD4+ T cells in P3a and P3b animals.
Since SHIV-E1 replicated to very high levels in immunodepleted RMs, we sought to determine if this passaged virus could replicate in immunocompetent hosts. Both P3a and P3b RMs had rapidly rising vRNA levels, with peaks of >108 vRNA copies/ml on day 14. Surprisingly, we noted a precipitous drop of CD4+ T cells to <200 cells/mm3, a pattern reminiscent of the acute pathogenicity of dual-tropic SHIV89.6P (14, 15). Indeed, in animal R555 (P3a), the CD4+ T cells never recovered. In the other recipient, R561 (P3b), there was a partial recovery of CD4+ T cells. Prompted by these unanticipated data, we examined the coreceptor usage of the virus isolated from passage 2, SHIV-E1p2d57. Indeed, this isolate turned CEMx174-GFP cells green, consistent with CXCR4 coreceptor usage (Fig. 4A). Next, we performed extended coreceptor assays in U87.CD4 and GHOST(3) cells expressing various individual human coreceptors. SHIV-E1p2d57 replicated in cells expressing either CCR5 or CXCR4 coreceptors (Fig. 4B), while the parental clone, SHIV-E1, infected only CCR5-expressing cells (Fig. 4B). Clearly, virus isolated at necropsy from the passage 2 animal had become dual tropic, in contrast to the parental SHIV-E1 infectious molecular clone (IMC), which used only CCR5.
The search for partially adapted SHIV-E1 with exclusive R5 tropism.
Given the unexpected coreceptor switch, we aimed to isolate R5-only virus from the latest time point possible during serial adaptation. To do this, we chose a two-pronged approach: (i) the generation of infectious molecular clones and (ii) the isolation of infectious, partially adapted virus by cocultivation with uninfected RM PBMC. The strategy to generate infectious env clones is depicted in Fig. 5A. Initially, env regions from the day 14 or day 57 isolate were cloned into the backbone of a proviral HIV clone, NL-LucR.T2A (16), to generate NL-LucR.E1p2d14 or NL-LucR.E1p2d57 IMCs, respectively.
Generation of the SHIV-E1p2 infectious molecular clone. (A) Steps involved in the generation of an exclusively R5 SHIV-E1p2 infectious molecular clone. (B) Virus production measured by a p24 ELISA of culture supernatants harvested on day 7 after exposure of U87.CD4.CCR5 or U87.CD4.CXCR4 cells to cell-free supernatants of 293T cells transfected with various NL-LucR.E1p2 IMCs. HIV-1NL4-3 and HIV-11084i were used as X4- and R5-tropic controls, respectively. (C) Infectivity on TZM-bl cells of serially diluted supernatants of 293T cells transfected with various IMCs of SHIV-E1p2. SHIV-KNH1144p4 (SHIV-A) (our unpublished data) was used as a positive control. (D) Ability of PBMC from 7 random naive rhesus macaque donors to support the replication of the SHIV-E1p2c183 IMC. The cell-free filtered supernatant of transfected 293T cells was assayed. Supernatants of IMC-exposed PBMC were collected on the days indicated.
Next, we examined the coreceptor usage of the different NL-LucR.E1p2 IMCs. Cells expressing either CCR5 or CXCR4 were incubated with virus, and p24 levels were measured from cell supernatants on day 7. All of the NL-LucR.E1p2d14 clones (clone 3 [c3], c7, c18, c20, and c21) replicated in CCR5-expressing cells but not in cells expressing CXCR4 (Fig. 5B). However, three of the four NL-LucR.E1p2d57 IMCs (c9, c23, and c26) replicated in both CCR5- and CXCR4-expressing cells (Fig. 5B). A single clone, c32, grew only in CXCR4-expressing cells (Fig. 5B). These data are consistent with the SHIV-E1p2d57 isolate containing strains that had switched coreceptor usage to become either dual tropic or X4-only tropic (c32). In contrast, NL-LucR.E1p2d14 IMCs were exclusively R5 tropic. Consequently, we used NL-LucR.E1p2d14 IMCs for further development. env regions of the latter were cloned into the backbone of SHIV-1157ipd3N4 (10) to generate SHIV-E1p2 IMCs (Fig. 5A). These proviral plasmids were transfected into 293T cells, followed by infectivity tests of filtered supernatants in TZM-bl cells; all SHIV-E1p2 IMCs were infectious, with SHIV-E1p2c183 showing the highest infectivity (Fig. 5C). SHIV-E1p2c183 replicated in PBMC from three of seven randomly selected naive RM donors (Fig. 5D). Compared to the parental clone, SHIV-E1, which had been unable to replicate in any RM PBMC previously, the partially adapted IMC SHIV-E1p2c183 replicated in 43% of randomly selected RM donor PBMC. These data indicate progress, albeit not yet full success, in adapting SHIV-E1 to the new host species.
P4 and P5 in immunocompetent animals.
Having successfully rescued partially adapted, cell-free virus as well as IMCs, we decided to combine all of these virus forms and use the successful “in vivo transfection” method again. Thus, the P4 animal, R798, received an i.m. inoculation of the IMC SHIV-E1p2c183 plus two i.v. inoculations of cell-free virus stocks of SHIV-E1p1d203 and SHIV-E1p2d14 on day 0 (Fig. 6A and andB).B). Two additional inoculations of the cell-free virus were given on subsequent days (Fig. 6B and andC,C, left). These combined inoculations resulted in high-level peak viremia of ∼108 vRNA copies/ml. Of note, this animal was not immunodepleted and thus had normal peripheral blood lymphocyte counts at the time of virus exposure. Over the course of the infection, the absolute number of CD4+ T cells dropped significantly. On day 28 after inoculation of P4 animal R798, infected blood was transferred to a new naive recipient, animal R974, again without immunodepletion. Viral titers approached 108 vRNA copies/ml, and viremia persisted (Fig. 6C, right). During acute viremia, the number of CD4+ T cells again dropped, reaching levels of <200 cells/mm3, indicating the pathogenicity of the passaged virus. Cell-free virus was reisolated from animal R974 on day 42 postinoculation and termed SHIV-E1p5 (Fig. 6A and andC).C). On day 332, this animal was euthanized due to reaching the study endpoint.
Serial passages P4 and P5 followed by isolation of SHIV-E1p5. (A) Animal R798 was inoculated with cell-free SHIV-E1p1d203 and SHIV-E1p2d14 i.v. and with SHIV-E1p2c183 IMC DNA by the i.m. route. Blood was transferred to animal R974 on day 28 after inoculation of animal R798. SHIV-E1p5 was isolated on day 42 after exposure of animal R974. Red boxes indicate R5-tropic virus. For P1 through P3a and P3b, see the legend to Fig. 2A. (B) Table summarizing the inocula, their sources, and routes of transfer during passages 4 and 5 of SHIV-E1. (C) Virus replication kinetics in animals R798 and R974. Red down arrow with asterisk indicates inoculation of RM R798 with proviral SHIV-E1p2c183 IMC DNA i.m. at two sites on day 0. Red down arrows (without asterisks) indicate inoculation of cell-free virus on three different days. Red up arrow for passage 5 for animal R974 indicates the reisolation of SHIV-E1p5 on day 42 after blood transfer, circles with P4 and P5 indicate the passage numbers, red boxes indicate R5-tropic virus, red lines with red circles indicate viral RNA copies per milliliter on a log scale, and axes on the right indicate absolute cell numbers (103).
SHIV-E1p5, an R5, mucosally transmissible, and pathogenic strain.
Next, we sought to ensure that the final SHIV-E1p5 isolate had retained its R5 tropism. We examined the coreceptor tropism of SHIV-E1p5 in CEMx174-GFP cells; no green fluorescence was observed, consistent with the lack of X4 coreceptor use (Fig. 7A). To confirm this finding, we also performed extended coreceptor assays. SHIV-E1p5 was able to infect cells expressing only the CCR5 coreceptor, indicating exclusive R5 use; SHIV-E1p5 specifically did not replicate in GPR15/BOB- and CXCR6/BONZO/STRL33-expressing cells (Fig. 7B).
SHIV-E1p5 is exclusively R5 tropic. (A) CEMx174-GFP cell infectivity assay with SHIV-E1p5 (i and iv), the X4-tropic positive control HIV-1NL4-3 (ii and v), and R5-tropic SHIV-1157ipd3N4 (iii and vi). (Top) Images acquired in bright field; (bottom) corresponding GFP fluorescence. (B) Infectivity of SHIV-E1p5 in U87.CD4 and GHOST.CD4 cells expressing various coreceptors. HIV-196USSN20 was used as a control.
To determine whether SHIV-E1p5 was well adapted to RM hosts, we examined its replication efficiency in vitro and in vivo. PBMC of six randomly selected naive RM donors were incubated with SHIV-E1 isolated from passage 1, passage 2, and passage 5. SHIV-E1p5 was able to replicate in all six RM donor PBMC, while SHIV-E1p1d203 and SHIV-E1p2d14 replicated only in two of six and four of six animals, respectively (Fig. 8A). An additional 10 random naive donors were chosen for additional screening of SHIV-E1p5 replication in RM PBMC. Eight out of 10 RM PBMC cultures supported SHIV-E1p5 replication (Fig. 8B). These results indicate that SHIV-E1p5 is better adapted for replication in RM PBMC than virus isolated from earlier passages. Finally, to assess mucosal transmissibility, a RM was challenged intrarectally (i.r.) with a single, high dose of SHIV-E1p5 (Fig. 8C) (1.4 × 105 50% tissue culture infective doses [TCID50]). This resulted in viremia of >108 vRNA copies/ml at week 2 postexposure, followed by persistent viremia (Fig. 8C). The number of CD4+ T cells dropped to <200 cells/mm3 by day 84 postinoculation, indicating pathogenicity (Fig. 8C). Clearly, SHIV-E1p5 is a mucosally transmissible, R5 clade E SHIV that depletes CD4+ T cells to low levels, consistent with AIDS.
SHIV-E1p5 has a tier 2 neutralization phenotype.
Next, we assessed the susceptibility of SHIV-E1p5 to a panel of anti-HIV mAbs as well as polyclonal serum or plasma samples in parallel with reference viruses, including tier 1 clade C SHIV-1157ipEL-p (17), its tier 2 counterpart SHIV-1157ipd3N4 (10), and tier 1A and tier 2 HIV CRF01_AE strains TH023.6 (18) and CNE55 (19) (Table 1). The polyclonal sera were standardized pools used to determine the neutralization sensitivity of primary HIV isolates. SHIV-E1p5 was not sensitive to neutralization by various Center for HIV/AIDS Vaccine Immunology (CHAVI) serum pools isolated from South African HIV-infected individuals (clade not determined). However, five of seven clade-matched plasma samples from HIV AE-infected individuals neutralized SHIV-E1p5 (Table 1, top red box), similar to that seen with CNE55. SHIV-E1p5 showed some neutralization by polyclonal anti-HIV clade C Abs in the HIVIG-C (HIV-specific IgG from HIV clade C-infected individuals) pool and soluble CD4 (sCD4) (Table 1, middle red box). While SHIV-E1p5 was relatively difficult to neutralize with known broadly neutralizing mAbs (nmAbs), it was sensitive to nmAbs targeting the membrane-proximal external region (MPER), and mAb LN01, a CD4 binding site-specific nmAb, neutralized SHIV-E1p5 (Table 1, middle and bottom red boxes). Overall, this neutralization profile is consistent with a tier 2 neutralization phenotype.
aResults are from SHIV-E1p5 prepared in human PBMC, consistent with the standard protocol that uses human serum/plasma and HIV isolates as reference strains.
bValues are the dilution for serum/plasma samples at which relative luminescence units (RLU) were reduced 50% (ID50).
cValues are the concentration for nmAbs or sCD4 at which RLU were reduced 50% (ID50).
dTier 2 clade C control.
eTier 1 clade C control.
fTier 1A CRF01_AE strain.
gTier 2 CRF01_AE strain.
hSerum samples from South African HIV+ individuals during chronic infection (infecting clade not determined). “Pool” refers to combining different bleed dates from the same individual.
iGeometric mean titer used to determine the neutralization tier compared to standard tier 1A, tier 1B, and tier 2 reference viruses. Tier cutoffs for AE plasma dilutions were >1,000 for tier 1A, 250 to 1,000 for tier 1B, 51 to 250 for tier 2, and ≤50 for tier 3.
env mutations arising during serial passage of SHIV-E1.
To examine the molecular evolution of the HIV-E envelope during SHIV-E1 adaptation, RNA isolated from the SHIV-E1p1d203, SHIV-E1p2d14, and SHIV-E1p5 isolates was analyzed by deep sequencing; nucleic acid mutations were used to predict amino acid changes (Fig.9). Predicted amino acid changes were compared to the envelope sequence of the parental clone, SHIV-E1. Of note, the entire evolution of SHIV-E1p2d14 occurred in the absence of selective pressure from host CD8+ cells and B cells due to double immunodepletion with cytotoxic mAbs. By the time when SHIV-E1p2d14 was isolated, the virus had replicated for a total of 67 days in the first two animals and represents the first intermediate isolate time-wise. Not only did the immunodepletion preclude the generation of anti-HIV Env Ab responses, but it also prevented adaptive cell-mediated immunity as well as innate immunity mediated by CD8+ NK cells. Therefore, mutations that arose in this early SHIV-E1 isolate (Fig.9,second panel) can be ascribed to the selection of viral quasispecies with improved replicative fitness in the new host environment, rather than antiviral host responses.
A number of mutations became fixed quickly, as indicated by the 100% prevalence in all sequence reads (Fig.9). One of the gp41 mutations, A790V, was found in all reads of the SHIV-E1p1d203 isolate. Interestingly, the prevalence of A790V in the passage 2 day 14 isolate, SHVI-E1p2d14, was only 65%. It should be kept in mind that virus isolated from passage 1 (SHIV-E1p1d203) had replicated for a total of 203 days in the first recipient animal. As mentioned above, SHIV-E1p2d14 had replicated for a total of 67 days in RMs, i.e., 53 days in the first animal until blood transfer and an additional 14 days in the second recipient. Consequently, more time had elapsed overall for SHIV-E1p1d203 (Fig. 9, top panel) than for SHIV-E1p2d14 (Fig. 9, second panel). In the final isolate, SHIV-E1p5, A790V was found at a prevalence of 100%. This mutation is located in the intracellular portion of gp41 that is not part of the HIV clade E insert. The same temporal pattern was also seen for I853T, located in the same gp41 region.
The pattern of predicted Env amino acid changes compared to the parental SHIV-E1 sequence was noticeably different in the last isolate, SHIV-E1p5. This is the only one of the four isolates shown in Fig. 9 where adaptive immune responses exerted their influence on Env evolution during serial passage. Interestingly, a number of low-level mutations appeared, especially in the ectodomain of gp41 (Fig. 9, bottom). Of note, SHIV-E1p5 is sensitive to neutralization by anti-MPER human nmAbs (Table 1, bottom red box). It is possible that the low-frequency mutations seen in gp41 represent early neutralization escape variants. Likewise, cell-mediated immunity could have participated in the selection of certain mutations. In this context, it is noteworthy that plasma of the passage 4 animal, R798, from the day of blood transfer to the last animal, R974, did not recognize HIV Env bands by Western blotting. Since SHIV-E1p5 was isolated from animal R974 on day 42, we sought to determine anti-Env Western blot reactivity on this day. Only a weak band was noted for gp160 but not for the other Env bands, indicating the beginning of host anti-Env antibody responses (data not shown).
Predicted amino acid changes in Env found exclusively in X4/dual-tropic SHIV-E1p2d57 quasispecies.
Next, we analyzed the predicted Env amino acid changes of the dual-tropic SHIV-E1p2d57 isolate by deep sequencing (Fig. 9, third panel) and compared these changes with those of the other three SHIV-E1 progeny. In parallel, we performed Sanger DNA sequencing of the V3 loop regions of various IMCs carrying clade E env (Fig. 10) since mutations in the V3 loop are known to be associated with a coreceptor switch; tropism was predicted by the PhenoSeq platform (20,–22) and compared to the experimental data (Fig. 4, ,5B,5B, ,7,7, and and10).10). Since HIV Env coreceptor switching has been reported for Thai individuals harboring HIV CRF01_AE strains (1, 12), we included Env sequences from such cases in our alignment (reference [Ref] clones) (Fig. 10).