Background
A major hindrance in drug, vaccine, and microbicide development for HIV/AIDS is limited utility of existing animal models. Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees. While chimpanzees can be productively infected, they are endangered, expensive, do not typically develop AIDS after HIV-1 infection, and their use in research engenders ethical concerns [
1]. This narrow host range of HIV-1 has compelled researchers to use macaque monkeys exposed or infected with SHIVenv, a chimera containing HIV-1 env coding regions within a simian immunodeficiency virus (SIV) backbone derived from rhesus macaques (
Macaca mulatta) infections. The genomic organization of SIVmac, HIV-1, and SHIV constructs are similar but encode for virus with significant functional variations. These differences are largely based on the accessory proteins, which appear to modulate viral replication in a host species-dependent manner impacting virus persistence, spread, and pathogenesis [
2‐
5]. A recent study showed that the HIV harboring the SIVmac vif gene could establish infection and was pathogenic in pigtailed macaques (
Macaca nemestrina) depleted for CD8+ T cells [
6].
SIV strains containing HIV-1 env genes (SHIVenv) have been successfully employed to infect macaques through intravenous and mucosal routes. Currently, most SHIVenv’s are clonal and even following propagation, do not contain a diverse representative of the HIV-1 population transmitted from donor to establish infection in a recipient with a single HIV-1 clone. Lack of diverse SHIVenv populations as innocula for macaque infection studies represents a resource gap for the rational development of HIV-1 vaccines and testing of microbicides. It is also critical to establish new env-based SHIVs for studies on pathogenesis and immune responses. However, the prospect of developing new infectious SHIVenv viruses is daunting considering the time consuming cloning procedures, the need for high titer virus propagation in extraneous cell lines, and the cost of testing infectivity in macaque infectivity using a reiterative SHIV strain-by-strain approach.
New HIV-1 infections (60–90%) originate from single HIV-1 variant or a limited number of transmitted/founder HIV-1 variants despite exposure to hundreds or thousands of HIV-1 clones from the donor partner [
7,
8]. This genetic bottleneck is less pronounced in individuals engaged in high-risk behaviors (anal-receptive intercourse or intravenous drug use) and in patients with ongoing sexually transmitted infections [
9]. Notably, acute infection with a “heterogeneous” infecting HIV-1 population has been linked to more rapid disease progression [
10]. As indicated above, most macaque models for primary HIV-1 infection involve exposure with only a single or highly homogeneous SHIVenv virus and do not reflect exposure to the highly heterogenous HIV-1 from donor to recipient. The HIV-1 clone(s) establishing primary infection in humans may have unique phenotypic properties from the inoculating HIV-1 population making it more apt for transmission. For example, transmitted virus preferentially employs CCR5 as co-receptor (R5 tropic) even though CXCR4 using HIV-1 may be present in the inoculating virus population. In the case of external pressure, use of preventative vaccines and microbicides should block new HIV-1 transmission. Prior to human trials, macaque models remain crucial for studies on HIV vaccine and microbicide testing [
11,
12]. However, few CCR5-using SHIVenv strains (e.g. SHIV
SF162, SHIV
CHN19, SHIV
1157ipd3N4) can maintain stable and prolonged infections [
13‐
15]. A recent study used a pool of SHIVenv viruses to infect macaques depleted of CD8+ T cells resulting identification of two pathogenic SHIVenv strains [
16]. Other studies suggested that the mutations in Env region could enhance macaque CD4-mediated entry and viral replication [
17,
18]. However, how these mutations impact SHIV pathogenesis in macaques has not been fully explored.
There were two main objectives for this study and both required the initial construction of SHIVs containing the transmitted subtype B
env genes, derived from the AHIs of CHAVI001 and other CHAVI clinical trials. The first objective was to identify R5 SHIVenv viruses with high transmission efficiency based on exposure of macaques to the heterogenous SHIVenv pool. As described herein, a single SHIVenv clone established macaque infection which prompted comprehensive genotypic and phenotypic analyses of why this SHIVenv was transmitted versus the other 15 in the pool. These analyses of transmission fitness required a battery of assays to measure proper virus assembly, replicative fitness, and the efficiency/kinetics of host cell entry, as well as transmission related modification (e.g. glycosylation) in these envelope proteins. The second objective in the companion article [
19] was to establish a pathogenic R5 SHIV from the SHIVenv with the highest transmission efficiency. It is important to stress that transmission efficiency and pathogenicity is likely related to different virus attributes. In past, the serial strain-by-strain cloning then testing in macaques has failed to identify a pathogenic R5 SHIVenv that provides a macaque model for prolonged HIV-1 infection in humans. Thus, we have serially passaged the highly transmissible SHIVenv to develop a new pathogenic R5 SHIVenv.
Discussion
Simian immunodeficiency viruses (SIV) or chimeras encoding the HIV-1 envelope (SHIVenv) are the most widely used in animal models of human HIV-1 infection [
38‐
41] but both have their shortcomings for HIV-1 prevention, vaccine, and pathogenesis studies. SIVmac was initially isolated from a macaque monkey in captivity who developed an AIDS-like disease [
38,
42] which prompted isolation of SIVmac
239, a clone that causes disease progression in these monkeys [
43,
44]. Subsequently, species-specific SIVs were isolated from various monkeys such as sooty mangabeys (SIVsmm) [
45], African green monkeys (SIVagm) [
46,
47] and mandrills (SIVmnd) [
48]. These SIVs establish asymptomatic, chronic infections with slow or no progression to disease in their natural hosts. It has been suggested that SIVmac emerged via a cross-species infection of the rhesus macaque with SIVsmm naturally found in sooty mangabeys [
45,
49]. SIVmac is similar to HIV-1 in genomic organization and somewhat similar in pathogenicity, e.g. depletion of CD4+ T cells in the gut early in disease [
50]. Both viruses can use CCR5 as a coreceptor and target CD4+ cells such as T-lymphocytes and macrophages, resulting in the complete loss of these cells but the disease course in macaques infected with SIVmac is short relative to that of HIV-1 infections (1–3 years versus 6–10 years). Infection of macaque monkeys with SIVmac is widely used as a model for HIV/AIDS to study disease progression and virus transmission but has significant limitation for prevention and vaccine studies.
With the results of the RV144 Thailand trial [
51‐
53] and the identification of broadly neutralizing antibodies, there is a renewed interest in humoral-based HIV vaccines as well as treatment with anti-HIV antibodies for both prevention and therapeutic use. Human trials with new vaccines or prevention strategies are typically preceded by safety and efficacy tests in macaque models. However, lack of diverse SHIVenv strains as challenge virus in macaques limits these vaccine and microbicide tests. Several vaccines tested with monovalent challenge SHIV did show complete efficacy in macaques but were less than optimal in human prevention trials [
54].
In this study, we generated a pool of 16 SHIVenv’s derived from the HIV-1
env genes of acute HIV-1 infections. The production of these SHIVenv’s was not a simple task and first required our development of an SIV and SHIV cloning system similar to the yeast-based cloning system for HIV-1 [
20,
55,
56]. Del Prete et al. [
16] used conventional cloning strategy to generate a cocktail of multiple of SHIVenv viruses. This method requires unique restriction enzyme sites which is usually difficult due to the extreme diversity of HIV sequences. In order to circumvent this difficulty, we utilized a yeast based recombination technology which only relies on sequence similarity and can insert a DNA fragment in one step within yeast without the need of restriction enzyme digestion, in vitro ligation and transformations. Through yeast recombination and gap repair, we shuttled over 70 versions of HIV-1
env and
gp120 coding regions from 20 AHI’s into our SHIV
KB9 vector and tested for virus production, entry efficiency, and virus propagation. Replacement of the HIV-1 KB9 with the entire
env coding region in SHIVenv resulted in very low levels of virus production. The
env gene carries the second exon of
tat/rev and generates a chimera of KB9/AHI Rev and Tat proteins which appears incompatible for efficient transcription and mRNA transport. To avoid the split in
rev/tat exons, we introduced the 20 AHI
env genes as cassettes that included the first exon of
tat/rev into SIVmac239 but again virus production and Env expression was too low to warrant further study. Previous studies have shown that the “KB9 Env” in the SIVmac239 has adapted to replication in macaques due in part to mutations in the extracellular domain of gp41 [
57], i.e. a region that would be replaced by rev/tat-env cassette. In the end, cloning of the gp120 and partial gp41 (upstream to the second exon of rev/tat) from these AHI’s into SHIV
KB9 preserved the KB9 Rev/Tat proteins and gp41 extracellular domain, and resulted in high level virus production from proviral transfections. These viruses (termed SHIVenv_B1 to _B20 for simplicity) had the correct stoichiometry of SIV proteins and fully functional HIV-1 glycoproteins but were unable to maintain long-term replication in various cell lines and primary, activated PBMCs of human or macaque origin.
SHIVenv_B viruses could support transient infection of different cell lines and primary macaque PBMCs but could not be propagated. Several groups have shown that propagation of SHIVenv in these primary cells or cell lines in not indicative of subsequent infection or pathogenesis in macaques [
13,
30,
31]. In fact, the original SHIVenv_89.6 clone did not replicate in culture but could infect macaques and is the progenitor of the CXCR4-using SHIVenv_KB9 virus. These observations were the primary reason to proceed with a pooled SHIVenv_B exposure in macaques. Based on Env function, viral protein content, and transient replication, we selected 16 of 20 SHIVenv_B viruses for this pool. Infection was observed in one of two macaques but only one of 16 SHIV viruses (i.e. SHIVenv_B3) successfully established infection. It is important to stress that the goal of this study was to establish prolonged macaque infection with SHIVenv derived from AHI and then passage through new macaques to enhance pathogenicity. We sequenced the entire SHIVenv_B genomes from each of the 16 clones and did not observe any mutations in the SHIV
KB9 backbone prior to infection. Furthermore, the SHIVenv_B3 during infection of m328-08 only had stochastic non-synonymous substitutions without dominant amino acid substitutions in the coding sequence across the entire proteome (including the env_B3). As reported in [
19], SHIVenv_B3 did establish a long term pathogenic infection when passaged from m328-08 to m165-05 but this enhanced virulence was associated with discrete amino acid substitution in Env_B3 appearing only in the m165-05 animal. Finally, we confirmed that SHIVenv_B3 alone could establish infection in three other macaques and did not require the presence of 15 other SHIVenv_B viruses in the pool.
Infection with only one of 16 SHIVenv_B clones could be due to a stochastic/random event or may be related to distinct properties of SHIVenv_B3 compared to the other AHI
env clones within the isogenic SHIV
KB9 backbone. Since the SHIVenv_B viruses could not replicate long term in culture, we constructed the equivalent the HIVenv_B chimeric viruses derived from the same 16 AHI
env genes but within an NL4-3 backbone. Despite the different SIV and HIV-1 backbones, we observed a direct correlation between the relative levels of cell fusion mediated 16 Env_B glycoproteins in the HIV-1 versus SIV backbone. A previous study involving macaque infections with a pool of SHIVenv viruses indicated that the SHIVenv with highest replication efficiency established infection in macaques [
16]. In both SHIVenv monoinfections the macaque PBMCs and in competitive fitness assays using human PBMCs and HIV-1env counterparts, the Env_B3 did not show enhanced replicative efficiency. The most fit Env_B16 was not detected in the infected macaque and neither was 12 other AHI Env’s that had higher replicative fitness and/or entry efficiency. The Env_B3 mediated fourth (of 16) highest level of host cell entry as determined by Veritrop. Cell fusion was with these 16 AHI Env_B glycoproteins as compared to cell fusion mediated with another set of 26 HIVenv chimeric viruses derived from AHI cohort at Aaron Diamond/New York University [
25], i.e. a pool of AHI that encompassed those SHIVenv employed by Del Prete et al. [
16]. When analyzing free virus using the Affinofile system [
24,
25], we found that the HIVenv chimeric viruses from CHAVI AHI and from the NYU AHI showed a wide range of affinity, avidity, and usage of CD4 and CCR5 for host cell entry. HIV-1 with Env_B3 was more efficient than the majority at host cell entry at high CCR5 across of range of CD4 levels on the cell surface but was only average when scavenging for low levels of CCR5. When comparing the kinetics of host cell entry, the HIVenv_B3 was slightly faster at these early steps of replication, ranking 3rd of the 16 HIVenv_B3 strains. However, HIVenv_B3 had lower than average replicative fitness when compared to the other 15 HIVenv_B viruses, all of which were competed against three control strains in human PBMCs. Our previous study with these HIVenv chimeric viruses also showed that HIVenv_B3 had no replicative advantage in primary macrophages, dendritic-T cell co-cultures, or infection of vaginal tissue [
37]. This array of phenotypic assays suggests that HIVenv_B3 is only average in terms of replicative fitness and host cell entry.
According to a recent study [
17], residue at position 375 is important in determining HIV-1 Env binding affinity to macaque CD4, i.e. S375 Env had minimum binding affinity, while mutation of S375M, Y, H, W, and F could significantly enhance the RhCD4 binding affinity and subsequent viral replication in macaque primary CD4 T cells. In our study, the 11 of 16 SHIV strains in the inoculating pool had an S375 which was associated with lower RhCD4 binding affinity. A375 and I375 were found in SHIVenv_B5 and _B12 While four strains had T375 (B16, B17 and B18). None of these residues at 375 were associated with high RhCD4 affinity. An earlier study by Boyd et al. [
18] reported that independent introduction of the A204E and G312V mutations yielded functional HIV-1 Env glycoproteins on SIV capable of mediating cell infection via huCD4 and RhCD4 receptors. None of our 16 strains had either 204E or 312V. In our study, we did not deplete macaque CD8+ T cells or alter the acute/early HIV-1
env genes cloned into the SHIVenv_KB9 backbone. Our intention was to select for the HIV-1
env gene (derived from acute infections) with the highest transmission efficiency in rhesus macaques and providing the best native HIV-1
env for subsequent SHIVenv studies on macaque infection and pathogenesis. It is also possible that in vitro adaptation step might help to find an effective SHIVenv strain.
Asmal et al. [
58] previously showed that there is a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. In the present study, the majority of 16 AHI viruses derived Envs including B3 contain histidine or other positively charged amino acids (e.g. Arginine) at position 12, and only B8 and B18 have non-basic amino acids at the same position. However, when comparing the Env sequences from these 16 AHIs, we did discover that the Env_B3 had the fewest conserved N-linked glycosylation sites in V1/V2 and V4/V5 loops and in
env gene overall. The Env_B3 also had the least positively charged V3 loop (aside from B7) and the lowest PSSM score, two measures predictive of relative CCR5 versus CXCR4 usage. Increased high-mannose N-linked glycosylation accounts for 50% of the Env glycoprotein mass, can reduce the viral antigenicity and protect functional regions of Env from host antibodies [
59‐
64]. Previous studies have shown that HIV-1 derived from acute/early infection typically have fewer N-linked glycosylation sites [
65,
66]. We propose that based on the further reduction of conserved N-linked sites, the Env_B3 may have 30% less high mannose glycans than even other AHI Env’s and up to 40% less than the average Env glycoprotein found during chronic disease. The role of Env glycosylation on HIV-1 transmission still remains unclear. Early reports suggested that high transmission efficiency of HIV-1 with reduced glycosylation was related to a compact Env glycoprotein where interactions with CD4/CCR5 and subsequent confirmation changes was not impeded by the bulk high mannose glycans. Since anti-Env antibodies do not appear for 2–3 weeks post infection, there would be no selection to maintain the highly glycosylated Env on the transmitted HIV-1 strains. However, we have no evidence to suggest an increased receptor binding, host cell entry, or replicative fitness of HIV-1 with fewer N-linked sites in Env. In contrast, the Env glycoproteins from chronic disease and with more N-linked sites typically have higher replicative fitness [
25]. Some SIV transmission studies showed that activated CD4 cells but not DCs are the initial targets of infection [
67‐
69]. It has been reported that reduced glycosylation of Env may enhance binding to α4β7 integrin found on gut CD4+ T cells, which may then be associated with rapid depletion of this Th17 T cells in the gut during early HIV-1 infection [
70]. On the other hand, glycans have been implicated in viral transmission through interaction with lectins, in particular the C-type lectin DC-SIGN, which is found on dendritic cells (DCs) and specific macrophages, and is thought to aid the transport of virus to anatomical sites rich in CD4+ T cells, such as lymph nodes [
71,
72]. The observation of transmitted HIV with fewer N-linked sites has always run counter to the role of DC sign in transmission. In addition, binding of HIV-1 to C-type lectin Langerin, found on the surface of Langerhans’ cells is now associated with endocytosis and degradation of HIV-1 [
71]. Langerhans’ cells are the primary DCs found in mucosal tissue. Regardless of these mechanisms, it is important to stress that selective transmission of the SHIVenv_B3, with the fewest N-linked sites, followed intravenous injection and not through exposure through a mucosal route. Thus, we can only speculate the role of various mechanisms on the selection of SHIVenv with the least glycosylated Env.