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Broadly neutralizing antibodies (bnAbs) against HIV-1 have been shown to protect from systemic infection. When employing a novel challenge virus that uses HIV-1 Env for entry into target cells during the first replication cycle, but then switches to SIV Env usage, we demonstrated that bnAbs also prevented mucosal infection of the first cells. However, it remained unclear whether antibody Fc-effector functions contribute to this sterilizing immunity. Therefore, additional challenge viruses were produced that contain SIV Env and graded doses of a fusion-defective trimer of HIV-1 Env, to which the bnAb, PGT121 can bind without interfering with the SIV Env-based cell entry. After administration of either PGT121 or its mutant deficient in Fc-effector functions, rhesus macaques were intrarectally exposed to these challenge viruses and to those using either HIV-1 Env or SIV Env for entry into the first cells. Both antibodies similarly reduced infection events with the challenge virus using HIV-1 Env by a factor close to 200. Incorporating fusion-defective HIV-1 Env trimers into the particles of the challenge viruses at densities observed in primary virus isolates did not reduce SIV Env-mediated infection events. The results indicate that the sparsity of bnAb binding-sites on HIV-1 virions limits the contribution of Fc-effector functions to provide sterilizing immunity against mucosal viral infection. Hence, harnessing Fc-effector functions for sterilizing immunity against mucosal HIV-1 infection may require strategies to increase the degree of antibody opsonization.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4.sup.+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4.sup.+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4.sup.+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4 + T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4 + T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N -glycans in SIV from CD4 + T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N -glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV’s vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N -glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human tumor virus with significant disease burden, particularly in Sub-Saharan Africa, where Kaposi’s sarcoma is prevalent. KSHV disproportionately affects immunocompromised individuals and men who have sex with men. A suitable animal model is critical for the development of a preventive vaccine, as it must replicate viral spread and immune interactions. The common marmoset (Callithrix jacchus) had previously been established as conducive to KSHV infection with non-BAC-derived virus via the intravenous (i.v.) route. We used this animal model to establish the ability to infect marmosets with bacterial artificial chromosome (BAC)-derived KSHV and to establish the role of individual glycoproteins (GPs) in the colonization of specific host tissues. We infected three groups of four animals each with BAC16-derived KSHV wild type (wt), KSHV gHASAELAAN, or KSHVΔK8.1. KSHV gHASAELAAN contains mutations in gH that interfere with binding to the EphA2 receptor, which normally forms a high-affinity complex with gH/gL, whereas KSHVΔK8.1 does not express GP K8.1. The KSHV gHASAELAAN mutant was used at a relatively higher concentration to overcome its intrinsic defect and to allow for analysis of tissue tropism. While seroconversion to the KSHV LANA antigen was variable, all animals exhibited clearly detectable viral DNA load in many tissues. Highest loads were detected in spleen, liver, heart and bone marrow. Viral DNA loads in these four tissues did not significantly differ between groups infected with KSHV wt or with the gHASAELAAN mutant. However, KSHVΔK8.1-infected animals exhibited significantly lower levels of viral DNA in liver and bone marrow, but not in spleen or heart. Analysis of immune cells demonstrated significant activation of CD4+ and CD8+ T-cells in KSHV wt-infected animals. In wt and gHASAELAAN-infected animals, different B cell subpopulations were found to expand after infection, which implies that the gH/gL glycoprotein complex may shape the host response to KSHV in unexpected ways. In summary, our findings demonstrate that neither the interaction with Eph family receptors nor GP K8.1 is essential for infection via the i.v. route but K8.1 plays a critical role for KSHV tissue tropism in vivo.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, the impact of these differences on viral spread and neutralization sensitivity remains largely unknown. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis revealed higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles differed between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.Competing Interest StatementThe authors have declared no competing interest.

Xenotransplantation using pig organs has achieved survival times of more than 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.