SHIV-C109p5 NHP induces rapid disease progression in elderly macaques with extensive GI viral replication.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirm the latency reversal properties of in vivo TGF-ß blockade, decrease viral reservoirs and stimulate immune responses. Treatment of eight female, SIV-infected macaques on ART with four 2-weeks cycles of galunisertib leads to viral reactivation as indicated by plasma viral load and immunoPET/CT with a 64Cu-DOTA-F(ab')2-p7D3-probe. Post-galunisertib, lymph nodes, gut and PBMC exhibit lower cell-associated (CA-)SIV DNA and lower intact pro-virus (PBMC). Galunisertib does not lead to systemic increase in inflammatory cytokines. High-dimensional cytometry, bulk, and single-cell (sc)RNAseq reveal a galunisertib-driven shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrate that galunisertib, a clinical stage TGF-ß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-ß blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on suppressive ART were treated with 4 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption(ATI). One macaque did not rebound, while the remaining rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with the 64Cu-DOTA-F(ab')2-p7D3-probe. Half to 1 Log decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk and single-cell RNAseq revealed a shift toward an effector phenotype in T and NK cells. In summary, we demonstrated that galunisertib, a clinical stage TGFß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine.

Despite the remarkable efficacy of COVID-19 vaccines, waning immunity and the emergence of SARS-CoV-2 variants such as Omicron represents a global health challenge. Here, we present data from a study in nonhuman primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine comprising the receptor binding domain of the ancestral strain (RBD-Wu) on the I53-50 nanoparticle adjuvanted with AS03, which was recently authorized for use in individuals 18 years or older. Vaccination induced neutralizing antibody (nAb) titers that were maintained at high concentrations for at least 1 year after two doses, with a pseudovirus nAb geometric mean titer (GMT) of 1978 and a live virus nAb GMT of 1331 against the ancestral strain but not against the Omicron BA.1 variant. However, a booster dose at 6 to 12 months with RBD-Wu or RBD-ß (RBD from the Beta variant) displayed on I53-50 elicited high neutralizing titers against the ancestral and Omicron variants. In addition, we observed persistent neutralization titers against a panel of sarbecoviruses, including SARS-CoV. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Vaccination resulted in protection against Omicron infection in the lung and suppression of viral burden in the nares at 6 weeks after the final booster immunization. Even at 6 months after vaccination, we observed protection in the lung and rapid control of virus in the nares. These results highlight the durable and cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine.

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.