The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4+ and CD8+ T cells, CD4+ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).

AbstractThe COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).

Similar to that of other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) lytic replication destroys the host cell, while the virus can persist in a latent state in synchrony with the host. During latency only a few genes are transcribed, and the question becomes one of what determines latent versus lytic gene expression. Here we undertake a detailed analysis of the latency-associated nuclear antigen (LANA [orf73]) promoter (LANAp). We characterized a minimal region that is necessary and sufficient to maintain high-level transcription in all tissues tested, including primary endothelial cells and B cells, which are the suspected natural host for KSHV. We show that in transient-transfection assays LANAp mimics the expression pattern observed for the authentic promoter in the context of the KSHV episome. Unlike other KSHV promoters tested thus far, LANAp is not affected by tetradecanoyl phorbol acetate or viral lytic cycle functions. It is, however, subject to control by LANA itself and cellular regulatory factors, such as p53. This is in contrast to the K14/vGCR (orf74) promoter, which overlaps LANAp and directs transcription on the opposite strand. We isolated a minimal cis-regulatory region sufficient for K14/vGCR promoter activity and show that it, too, mimics the regulation observed for the authentic viral promoter. In particular, we demonstrate that its activity is absolutely dependent on the immediate-early transactivator orf50, the KSHV homolog of the Epstein-Barr virus Rta transactivator.Using representational difference analysis Chang et al. (6) demonstrated the presence of a novel human virus in Kaposi's sarcoma (KS) biopsy samples: Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8. KSHV has since been detected in all manifestations of KS as well as in two lymphoproliferative disorders: primary effusion lymphoma (4) and multicentric Castleman's disease (53). On the basis of the complete sequence of the 137-kbp double-stranded DNA genome, KSHV is classified as a gamma-2 herpesvirus, a member of the lymphotropic subgroup of the Herpesviridae (17,36,45).The epidemiological evidence implicating KSHV as a causative agent for KS is compelling (reviewed in reference 51). (i) KSHV DNA is found in Ͼ90% of KS biopsy samples. (ii) KSHV latent mRNAs and proteins are detectable in every KS spindle cell by in situ methods. (iii) Antibodies to KSHV exist in Ն80% of KS patients, and multiple viral antigens have been identified as targets of this response. (iv) Increases in peripheral-blood viral load as well as anti-KSHV antibody titer precede the onset of disease and correlate with increased risk for KS. These observations establish KSHV as a necessary cofactor for KS.KSHV, like all herpesviruses, displays two modes of replication: lytic replication, during which the host cell is destroyed and viral progeny are released, and latent replication, during which the viral genome persists indefinitely and no viral progeny are released. In KS, KSHV persists latently in Ն90% of...

In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.

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.

Certain lymphomas in AIDS patients, such as primary effusion lymphoma (PEL), are closely associated with the lymphotropic ␥ herpes virus Kaposi's sarcoma-associated herpes virus (KSHV), also called human herpesvirus 8. The virus is thought to be essential for tumorigenesis, yet systems to investigate PEL in vivo are rare. Here we describe PEL tumorigenesis in a new xenograft model. Embedded in Matrigel, PEL cells formed rapid, well-organized, and angiogenic tumors after s.c. implantation of C.B.17 SCID mice. Without Matrigel we did not observe comparable tumors, which implies that extracellular support and/or signaling aids PEL. All of the tumors maintained the KSHV genome, and the KSHV latent protein LANA/orf73 was uniformly expressed. However, the expression profile for key lytic mRNAs, as well as LANA-2/vIRF3, differed between tissue culture and sites of implantation. We did not observe a net effect of ganciclovir on PEL growth in culture or as xenograft. These findings underscore the importance of the microenvironment for PEL tumorigenesis and simplify the preclinical evaluation of potential anticancer agents.

SummaryThe resurgence of pertussis may originate from inefficient vaccine-mediated protection against infection by Bordetella pertussis. Here, we show that the live attenuated vaccine BPZE1 is safe in nonhuman primates and induces robust protection against both B. pertussis disease and infection.