Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

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Vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus

Andes virus (ANDV) is a highly pathogenic South American hantavirus that causes hantavirus pulmonary syndrome (HPS). A high case fatality rate, the potential for human-to-human transmission, the capacity to infect via aerosolization, and the absence of effective therapies make it imperative that a safe, fast-acting, and effective ANDV vaccine be developed. We generated and characterized a recombinant vesicular stomatitis virus (VSV) vector expressing the ANDV surface glycoprotein precursor (VSVΔG/ANDVGPC) as a possible vaccine candidate and tested its efficacy in the only lethal-disease animal model of HPS. Syrian hamsters immunized with a single injection of VSVΔG/ANDVGPC were fully protected against disease when challenged at 28, 14, 7, or 3 days postimmunization with a lethal dose of ANDV; however, the mechanism of protection seems to differ depending on when the immunization occurs. At 28 days postimmunization, a lack of detectable ANDV RNA in lung, liver, and blood tissue samples, as well as a lack of seroconversion to the ANDV nucleocapsidprotein in nearly all animals, suggested largely sterile immunity. The vaccine was able to generate high levels of neutralizing anti-ANDV G(N)/G(C) antibodies, which seem to play a role as a mechanism of vaccine protection. Administration of the vaccine at 7 or 3 days before challenge also resulted in full protection but with no specific neutralizing humoral immune response, suggesting a possible role of innate responses in protection against challenge virus replication. Administration of the vaccine 24 h postchallenge was successful in protecting 90% of hamsters and again suggested the induction of a potent antiviral state by the recombinant vector as a potential mechanism. Overall, our data suggest the potential for the use of the VSV platform as a fast-acting and effective prophylaxis/postexposure treatment against lethal hantavirus infections.     

Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques

The primary cause of severe acute respiratory syndrome (SARS) is a newly discovered coronavirus. Replication of this SARS coronavirus (SCV) occurs mainly in the lower respiratory tract, and causes diffuse alveolar damage. Lack of understanding of the pathogenesis of SARS has prevented the rational development of a therapy against this disease. Here we show extensive SCV antigen expression in type 1 pneumocytes of experimentally infected cynomolgus macaques (Macaca fascicularis) at 4 d postinfection (d.p.i.), indicating that this cell type is the primary target for SCV infection early in the disease, and explaining the subsequent pulmonary damage. We also show that prophylactic treatment of SCV-infected macaques with the antiviral agent pegylated interferon-alpha (IFN-alpha) significantly reduces viral replication and excretion, viral antigen expression by type 1 pneumocytes and pulmonary damage, compared with untreated macaques. Postexposure treatment with pegylated IFN-alpha yielded intermediate results. We therefore suggest that pegylated IFN-alpha protects type 1 pneumocytes from SCV infection, and should be considered a candidate drug for SARS therapy.     

Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.     

Virus load and sequence variation in simian retrovirus type 2 infection

The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome.     

Maternal transmission of type D simian retrovirus (SRV-2) in pigtailed macaques

Pregnant macaques were used as a natural model for maternal-infant transmission of SRV-2 retrovirus. Fifty-one pregnant females were placed into one of four virus/antibody groups. Nonviremic mothers produced 100% virus-negative offspring at birth. In contrast, viremic mothers produced offspring which were 17% virus-negative and 83% virus-positive at birth. SRV-2 infection occurred principally in utero by the transplacental route. Infants born to viremic mothers exhibited low birth weight, prematurity, high perinatal death, and increased incidence of SAIDS.     

Relationship of mitogen reactivity to type D retrovirus infection in Celebes black macaques (Macaca nigra)

The Celebes black macaque (Macaca nigra) colony at the Oregon Regional Primate Research Center has a high incidence of an immunodeficiency syndrome characterized by recurrent diarrhea and the development of retroperitoneal fibromatosis (RF). We have examined the relationship of type D viral infection to the immunodeficiency syndrome by surveying the colony for viral infection and for mitogen reactivity. Type D virus-positive monkeys (28% of the colony) have a higher prevalence of diarrhea, splenomegaly, lymphadenopathy and weight loss than do virus-negative monkeys, and RF has been found to occur only in virus-positive animals. Comparison of the concanavalin A (con-A) and phytohemagglutinin reactivities of the virus-positive and -negative populations has revealed no significant difference. However, within the virus-positive population, those with RF have reduced con-A reactivity and there are both high and low mitogen responders in the groups lacking RF. Thirty-two percent of the virus-positive monkeys are free of clinical symptoms, 40% have clinical symptoms but no RF, and 27% have clinical symptoms and RF. Five of the six monkeys with RF are older than the RF-free monkeys but monkeys are susceptible to type D retrovirus infection regardless of age or sex. The progressive nature of this immunodeficiency syndrome, its broad age range, and the probability that the etiological agent is also a type D retrovirus and the similarity of RF to Kaposi's sarcoma make this a potentially useful model for human AIDS.     

Association of SAIDS/RF-related signs with current or past SAIDS type 2 retrovirus infection in a colony of Celebes black macaques

The 83 members of the Celebes black macaque (Macaca nigra) colony were screened for viremia with simian acquired immunodeficiency syndrome (SAIDS) type 2 retrovirus and antibodies against the retrovirus. On the basis of this screening, the Celebes colony was divided into four groups: retrovirus-positive/seropositive (virus+/Ab+); retrovirus-negative/seropositive (virus-/Ab+); retrovirus-positive/seronegative (virus+/Ab-); and retrovirus-negative/seronegative (virus-/Ab-). Monkeys in the virus+/Ab+ group displayed more major clinical signs and required medication more times than monkeys in the other groups. In contrast, monkeys in the virus-/Ab- group had fewer health problems than monkeys in the other groups. The five monkeys that had surgically confirmed retroperitoneal fibromatosis (RF), palpable abdominal masses, or both, were in the virus+/Ab+ group. Some of the monkeys in groups with current or past retrovirus infection were well clinically. There were no statistically significant differences in the mitogen reactivities of mononuclear cells obtained from monkeys of the different groups.     

Isolation of a type D retrovirus from B-cell lymphomas of a patient with AIDS.

An atypical syncytial variant of a high-grade Burkitt's-type B-cell lymphoma from a patient with AIDS who was seropositive for human immunodeficiency virus type 1 was studied. A productive type D retrovirus infection was identified in early-passage cell lines derived from two lymphomas from this patient. Nucleotide and amino acid sequence analysis as well as immunologic reactivity indicated that the isolated virus was highly related to Mason-Pfizer monkey virus (MPMV). MPMV is an immunosuppressive type D retrovirus that causes an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by polymerase chain reaction generated the appropriate MPMV-specific fragments and indicated that the patient was infected with the MPMV-like retrovirus. In addition, the patient's serum contained antibodies which recognized type D viral env proteins (gp70 and gp20) and gag proteins (p27 and p14). Although there have been reports of human cell lines infected with type D retroviruses and of type D-reactive human sera, this is the first evidence of a type D retrovirus infection in a human confirmed by virus isolation, serum reactivity, and viral DNA identification in tumor tissue.     

Serologic and virologic analysis of type D simian retrovirus infection in a colony of Celebes black macaques (Macaca nigra)

Celebes macaques were tested for type D simian retrovirus (SRV) infection. SRV infection was first detected in one serum sample collected during 1980. By 1983, 32 of 46 monkeys (70%) were infected. Serotyping of the SRV isolates determined that 0/26 of the isolates were SRV-1; 24/26 were SRV-2; 1/26 was SRV-5; and 1/26 could not be typed. Restriction endonuclease mapping confirmed the SRV-2C and SRV-5 isolates. In addition, two SRV-2C variants were detected.     

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus

BackgroundZika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV.Methodology/Principle findingsWe have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge.Conclusions/SignificanceThese studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.     

Codelivery of the chemokine CCL3 by an adenovirus-based vaccine improves protection from retrovirus infection

Processing and presentation of vaccine antigens by professional antigen-presenting cells (APCs) is of great importance for the efficient induction of protective immunity. We analyzed whether the efficacy of an adenovirus-based retroviral vaccine can be enhanced by coadministration of adenovirus-encoded chemokines that attract and stimulate APCs. In the Friend retrovirus (FV) mouse model we coexpressed CCL3, CCL20, CCL21, or CXCL14 from adenoviral vectors, together with FV Gag and Env antigens, and then analyzed immune responses and protection from pathogenic FV infection. Although most tested chemokines did not improve protection against FV challenge, mice that received adenoviral vectors encoding CCL3 together with FV antigens showed significantly better control over viral loads and FV-induced disease than mice immunized with the viral antigens only. Improved protection correlated with enhanced virus-specific CD4+ T cell responses and higher neutralizing antibody titers. To apply these results to an HIV vaccine, mice were immunized with adenoviral vectors encoding the HIV antigens Env and Gag-Pol and coadministered vectors encoding CCL3. Again, this combination vaccine induced higher virus-specific antibody titers and CD4+ T cell responses than did the HIV antigens alone. These results indicate that coexpression of the chemokine CCL3 by adenovirus-based vectors may be a promising tool to improve antiretroviral vaccination strategies.     

The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection

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Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer.     

Elimination of type D retrovirus infection from group-housed rhesus monkeys using serial testing and removal.

Type D retrovirus was successfully eliminated from an infected population of group-housed rhesus monkeys by serial testing of all animals for virus and antibody and subsequent removal of positives. This population of 53 rhesus had been housed together for 1 year prior to the initiation of the test and removal program, with six deaths from type D retrovirus-induced immunodeficiency disease occurring during this period. No new infections were detected after four rounds of testing. Of the 47 animals present at the start of the testing program, 17 (35%) remained after the elimination of type D virus from this group. These animals and their offspring have remained healthy and antibody negative for more than 2 years. These results demonstrate that elimination of type D retroviruses from rhesus macaque colonies is feasible, and that the objective of establishing and maintaining retrovirus-free colonies is realistic and achievable.     

Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques

We evaluated three nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of four rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, enzyme-linked immunosorbent assay (ELISA) antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge.     

Vesicular stomatitis virus evolution during alternation between persistent infection in insect cells and acute infection in mammalian cells is dominated by the persistence phase

Vesicular stomatitis virus has the potential for very rapid evolution in the laboratory, but like many other arboviruses, it evolves at a relatively slow rate in the natural environment. Previous work showed that alternating replication in different cell types does not promote stasis. In order to determine whether other factors promote stasis, we compared the fitness trajectories of populations evolving during acute infections in mammalian cells, populations evolving during persistent infections in insect cells, and populations evolving during alternating acute and persistent infection cycles. Populations evolving under constant conditions increased in fitness in the environment in which they replicated. An asymmetric trade-off was observed such that acute infection had no cost for persistence but persistent replication had a dramatic cost for acute infection in mammalian cells. After an initial period of increase, fitness remained approximately constant in all the populations that included persistent replication, but fitness continuously increased in populations evolving during acute infections. Determination of the consensus sequence of the genes encoding the N, P, M, and G proteins showed that the pattern of mutation accumulation was coherent with fitness changes during persistence so that once fitness reached a maximum, the rate of mutation accumulation dropped. Persistent replication dominated both the genetic and the phenotypic evolution of the populations that alternated between acute infection of mammalian cells and persistence in insect cells, and fitness loss was observed in the mammalian environment despite periodic replication in mammalian cells. These results show that stasis can be achieved without good levels of adaptation to both the mammalian and the insect environments.