Genome-wide real-time PCR for West Nile virus reduces the false-negative rate and facilitates new strain discovery

West-Nile virus (WNV) causes significant morbidity and mortality worldwide. Transplant and transfusion recipients as well as the elderly are particularly at risk. WNV shows strain variation from season to season and from locale to locale. This poses a significant problem for diagnosis. Most assays use a single primer pair to detect WNV by QPCR, and can fail to detect novel stains. To overcome this limitation, a genome-wide, multiple primer-based real-time QPCR assay was developed for WNV. The same assay can be used for quantitation, viral variant discovery as well as for amplification of the entire viral genome using a single annealing temperature. It improves upon routine diagnosis as well as facilitates laboratory investigations of the pathology of WNV

Immune features that afford protection from clinical disease versus sterilizing immunity to Bordetella pertussis infection in a nonhuman primate model of whooping cough

The respiratory bacterial infection caused by Bordetella pertussis (whooping cough) is the only vaccine-preventable disease whose incidence has been increasing over the last 3 decades. To better understand the resurgence of this infection, a baboon animal model of pertussis infection has been developed. Naïve baboons that recover from experimental pertussis infection are resistant both to clinical disease and to airway colonization when re-challenged. In contrast, animals vaccinated with acellular pertussis vaccine and experimentally challenged do not develop disease, but airways remain colonized for 4-6 weeks. We explored the possibility that the IgG antibody response to pertussis infection is qualitatively different from antibodies induced by acellular pertussis vaccination. IgG was purified from pertussis-convalescent baboons shown to be resistant to pertussis disease and airway colonization. Purified IgG contained high titers to pertussis toxin, pertactin, and filamentous hemagglutinin. This pertussis-immune IgG or control IgG was passively transferred to naïve, juvenile baboons before experimental airway pertussis inoculation. The control animal that received normal IgG developed a typical symptomatic infection including leukocytosis, cough and airway colonization for 4 weeks. In contrast, baboons that received convalescent IgG maintained normal WBC counts and were asymptomatic. However, despite remaining asymptomatic, their airways were colonized for 4-6 weeks with B. pertussis. All animals developed IgG and IgA anti-pertussis antibody responses. Interestingly, the clearance of B. pertussis from airways coincided with the emergence of a serum anti-pertussis IgA response. These studies demonstrate that passive administration of pertussis-specific IgG from previously infected animals can prevent clinical disease but does not affect prolonged airway colonization with B. pertussis. This outcome is similar to that observed following acellular pertussis vaccination. Understanding immune mechanisms—other than IgG—that are capable of preventing airway colonization with B. pertussis will be critical for developing more effective vaccines to prevent whooping cough

Fifteen Years of Sm-p80-Based Vaccine Trials in Nonhuman Primates: Antibodies From Vaccinated Baboons Confer Protection in vivo and in vitro From Schistosoma mansoni and Identification of Putative Correlative Markers of Protection.

Recent advances in systems biology have shifted vaccine development from a largely trial-and-error approach to an approach that promote rational design through the search for immune signatures and predictive correlates of protection. These advances will doubtlessly accelerate the development of a vaccine for schistosomiasis, a neglected tropical disease that currently affects over 250 million people. For over 15 years and with contributions of over 120 people, we have endeavored to test and optimize Sm-p80-based vaccines in the non-human primate model of schistosomiasis. Using RNA-sequencing on eight different Sm-p80-based vaccine strategies, we sought to elucidate immune signatures correlated with experimental protective efficacy. Furthermore, we aimed to explore the role of antibodies through in vivo passive transfer of IgG obtained from immunized baboons and in vitro killing of schistosomula using Sm-p80-specific antibodies. We report that passive transfer of IgG from Sm-p80-immunized baboons led to significant worm burden reduction, egg reduction in liver, and reduced egg hatching percentages from tissues in mice compared to controls. In addition, we observed that sera from Sm-p80-immunized baboons were able to kill a significant percent of schistosomula and that this effect was complement-dependent. While we did not find a universal signature of immunity, the large datasets generated by this study will serve as a substantial resource for further efforts to develop vaccine or therapeutics for schistosomiasis

In vitro and In vivo Susceptibility of Baboons (Papio sp.) to Infection with and Apparent Antibody Reactivity to Simian Betaretrovirus (SRV).

Despite the lack of confirmed reports of an exogenous Simian betaretrovirus (SRV) isolated from baboons (Papio sp.), reports of simian endogenous gammaretrovirus (SERV) in baboons with complete genomes suggest that such viruses may be potentially infectious. In addition, serologic tests have repeatedly demonstrated antibody reactivity to SRV in baboons from multiple colonies. These findings complicate the management and use of such animals for research. To provide further insight into this situation, we performed in vitro and in vivo studies to determine if baboons are or can be infected with SRV. In our initial experiment, we were not able to isolate SRV from 6 seropositive or sero-indeterminate baboons by coculturing their peripheral blood mononuclear cells (PBMC) with macaque PBMC or permissive cell lines. In a subsequent experiment, we found that baboon PBMC infected in vitro with high dose SRV were permissive to virus replication. To test in vivo infectibil- ity, groups of naive baboons were infused intravenously with either (i) the same SRV tissue culture virus stocks used for the in vitro studies, (ii) SRV antibody positive and PCR positive macaque blood, (iii) SRV antibody positive or indeterminate, but PCR negative baboon blood, or (iv) SRV antibody and PCR negative baboon blood. Sustained SRV infection, as defined by reproducible PCR detection and/or antibody seroconversion, was confirmed in 2 of 3 baboons receiving tissue culture virus but not in any recipients of transfused blood from seropositive macaques or baboons. In conclusion, the data indicate that even though baboon cells can be infected experimentally with high doses of tissue culture grown SRV, baboons that are repeatedly SRV antibody positive and PCR negative are unlikely to be infected with exogenous SRV and thus are unlikely to transmit a virus that would threaten the SPF status of captive baboon colonies

Zika virus infection at mid-gestation results in fetal cerebral cortical injury and fetal death in the olive baboon.

Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to as Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and non-human primates (NHPs). In macaques, fetal CZS outcomes from maternal ZIKV infection range from none to significant. In the present study we develop the olive baboon (Papio anubis), as a model for vertical transfer of ZIKV during pregnancy. Four mid-gestation, timed-pregnant baboons were inoculated with the French Polynesian ZIKV isolate (104 ffu). This study specifically focused on the acute phase of vertical transfer. Dams were terminated at 7 days post infection (dpi; n = 1), 14 dpi (n = 2) and 21 dpi (n = 1). All dams exhibited mild to moderate rash and conjunctivitis. Viremia peaked at 5-7 dpi with only one of three dams remaining mildly viremic at 14 dpi. An anti-ZIKV IgM response was observed by 14 dpi in all three dams studied to this stage, and two dams developed a neutralizing IgG response by either 14 dpi or 21 dpi, the latter included transfer of the IgG to the fetus (cord blood). A systemic inflammatory response (increased IL2, IL6, IL7, IL15, IL16) was observed in three of four dams. Vertical transfer of ZIKV to the placenta was observed in three pregnancies (n = 2 at 14 dpi and n = 1 at 21 dpi) and ZIKV was detected in fetal tissues in two pregnancies: one associated with fetal death at ~14 dpi, and the other in a viable fetus at 21 dpi. ZIKV RNA was detected in the fetal cerebral cortex and other tissues of both of these fetuses. In the fetus studied at 21 dpi with vertical transfer of virus to the CNS, the frontal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, disorganized migration of immature neurons to the cortical layers, and signs of pathology in immature oligodendrocytes. In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL6 expression. Of interest, in one fetus examined at 14 dpi without detection of ZIKV RNA in brain and other fetal tissues, increased neuroinflammation (IL6 and microglia) was observed in the cortex. Although the placenta of the 14 dpi dam with fetal death showed considerable pathology, only minor pathology was noted in the other three placentas. ZIKV was detected immunohistochemically in two placentas (14 dpi) and one placenta at 21 dpi but not at 7 dpi. This is the first study to examine the early events of vertical transfer of ZIKV in a NHP infected at mid-gestation. The baboon thus represents an additional NHP as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air–liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals