High Titers Of Virus-neutralizing Antibodies Research Articles

A rationally designed combined intranasal adjuvant drives robust and broad humoral, cellular, and mucosal immune responses to SARS-CoV-2

Abstract Multiple FDA-approved SARS-CoV-2 vaccines have provided excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Furthermore, while these parenteral vaccines have provided substantial protection, they have been less effective in inducing mucosal immune responses which are critical for effectively blocking viral transmission in vaccinated individuals. As mucosal immune responses are most effectively induced through infection or mucosal immunization, we have developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). Through activating this array of receptors, we have shown that the NE/IVT adjuvant better recapitulates the innate immune activation events that occur during natural viral infection, subsequently leading to more tailored and effective downstream adaptive immune responses. The combination adjuvant with various spike protein antigens elicited robust protective humoral, mucosal, and cellular responses to SARS-CoV-2 in mice, with markedly enhanced T H1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous and antigenically distant drift variants of SARS-CoV-2. These results highlight the potential of this adjuvant to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates for use mucosally. This research was supported by R01-AI160706 to P.W. and M.S, and in part by R01-DK130425 to M.S.

Longitudinal analysis of SARS-CoV-2 reinfection reveals distinct kinetics and emergence of cross-neutralizing antibodies to variants of concern.

The ongoing evolution of SARS-CoV-2 continues to raise new questions regarding the duration of immunity to reinfection with emerging variants. To address these knowledge gaps, controlled investigations in established animal models are needed to assess duration of immunity induced by each SARS-CoV-2 lineage and precisely evaluate the extent of cross-reactivity and cross-protection afforded. Using the Syrian hamster model, we specifically investigated duration of infection acquired immunity to SARS-CoV-2 ancestral Wuhan strain over 12 months. Plasma spike- and RBD-specific IgG titers against ancestral SARS-CoV-2 peaked at 4 months post-infection and showed a modest decline by 12 months. Similar kinetics were observed with plasma virus neutralizing antibody titers which peaked at 2 months post-infection and showed a modest decline by 12 months. Reinfection with ancestral SARS-CoV-2 at regular intervals demonstrated that prior infection provides long-lasting immunity as hamsters were protected against severe disease when rechallenged at 2, 4, 6, and 12 months after primary infection, and this coincided with the induction of high virus neutralizing antibody titers. Cross-neutralizing antibody titers against the B.1.617.2 variant (Delta) progressively waned in blood over 12 months, however, re-infection boosted these titers to levels equivalent to ancestral SARS-CoV-2. Conversely, cross-neutralizing antibodies to the BA.1 variant (Omicron) were virtually undetectable at all time-points after primary infection and were only detected following reinfection at 6 and 12 months. Collectively, these data demonstrate that infection with ancestral SARS-CoV-2 strains generates antibody responses that continue to evolve long after resolution of infection with distinct kinetics and emergence of cross-reactive and cross-neutralizing antibodies to Delta and Omicron variants and their specific spike antigens.

Pathological Evaluation of Porcine Circovirus 2d (PCV2d) Strain and Comparative Evaluation of PCV2d and PCV2b Inactivated Vaccines against PCV2d Infection in a Specific Pathogen-Free (SPF) Yucatan Miniature Pig Model.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine.

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection as drift variants continue to emerge. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

Virus-Like Particles Derived From a Virulent Strain of Pest des Petits Ruminants Virus Elicit a More Vigorous Immune Response in Mice and Small Ruminants Than Those From a Vaccine Strain

Peste des petits ruminants (PPRs) is highly contagious, acute or subacute disease of small ruminants caused by peste des petits ruminants virus (PPRV). To date, several studies have designed and evaluated PPRV-like particles (VLPs) as a vaccine candidate for the prevention and control of PPR, with the majority of these VLPs constructed using sequences derived from a PPRV vaccine strain due to its high immunogenicity. However, because of the lack of available genetic material and certain structural proteins and/or the alteration of posttranslational glycosylation modifications, the immunogenicity of VLPs derived from a vaccine strain is not always optimal. In this study, two PPRV VLP candidates, derived from either the lineage IV Tibet/30 virulent strain or the lineage II Nigeria 75/1 vaccine strain, were generated using a baculovirus system through the coexpression of the PPRV matrix (M), hemagglutinin (H), and fusion (F) proteins in the high expression level cell line High Five. These VLPs were then used to immunize mice, goats, and sheep followed by two boosts after primary immunization. Both VLPs were found to induce a potent humoral immune response as demonstrated by the high ratio of immunoglobulin G1 (IgG1) to IgG2a. In all animals, both VLPs induced high titers of virus-neutralizing antibodies (VNAs), as well as H- and F-specific antibodies, with the Tibet/30 VLPs yielding higher antibody titers by comparison to the Nigeria 75/1 VLPs. Studies in mice also demonstrated that the Tibet/30 VLPs induced a more robust interleukin 4 and interferon γ response than the Nigeria 75/1 VLPs. Goats and sheep immunized with both VLPs exhibited a robust humoral and cell-mediated immune response. Furthermore, our results demonstrated that the VLPs derived from the virulent lineage IV Tibet/30 strain were more immunogenic, inducing a more potent and robust humoral and cell-mediated immune response in vaccinated animals by comparison to the lineage II Nigeria 75/1 vaccine strain VLPs. In addition, VNA titers were significantly higher among animals vaccinated with the Tibet/30 VLPs by comparison to the Nigeria 75/1 VLPs. Taken together, these findings suggest that VLPs derived from the virulent lineage IV Tibet/30 strain are more immunogenic by comparison to those derived from the lineage II Nigeria 75/1 vaccine strain and thus represent a promising vaccine candidate for the control and eradication of PPR.

Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China.

Porcine deltacoronavirus (PDCoV) was first detected in Hong Kong and has recently spread to many countries around the world. PDCoV causes acute diarrhea and vomiting in pigs, resulting in significant economic losses in the global pork industry. In this study, a Chinese PDCoV strain, designated CHN-HG-2017, was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central China. Subsequently, the virus was identified by an indirect immunofluorescence assay and electron microscopy. A nucleotide sequence alignment showed that the whole genome of CHN-HG-2017 is 97.6%-99.1% identical to other PDCoV strains. Analysis of potential recombination sites showed that CHN-HG-2017 is a possible recombinant originating from the strains CH/SXD1/2015 and Vietnam/HaNoi6/2015. Furthermore, the pathogenicity of this recombinant PDCoV strain was investigated in 5-day-old piglets by oral inoculation. The challenged piglets developed typical symptoms, such as vomiting, anorexia, diarrhea and lethargy, from 1 to 7 days post-inoculation (DPI). Viral shedding was detected in rectal swabs until 14 DPI in the challenged piglets. Interestingly, high titers of virus-neutralizing antibodies in sera were detected at 21 DPI. Tissues of small intestines from CHN-HG-2017-infected piglets at 4 DPI displayed significant macroscopic and microscopic lesions with clear viral antigen expression. Our analysis of the full genome sequence of a recombinant PDCoV and its virulence in suckling piglets might provide new insights into the pathogenesis of PDCoV and facilitate further investigation of this newly emerged pathogen.

Zika virus-like particle (VLP) based vaccine.

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

Immunization with Low Doses of Recombinant Postfusion or Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the Cotton Rat Model Independently of the Presence of a Th1-Biasing (GLA-SE) or Th2-Biasing (Alum) Adjuvant.

Respiratory syncytial virus (RSV) infection of children previously immunized with a nonlive, formalin-inactivated (FI)-RSV vaccine has been associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of nonlive RSV vaccines targeting RSV-naive children and infants. The fusion glycoprotein (F) of RSV in either its postfusion (post-F) or prefusion (pre-F) conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here, we report the evaluation of RSV post-F and pre-F in combination with glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE) and alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus-neutralizing antibodies and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant at levels comparable to FI-RSV-immunized controls. This occurred despite neutralizing-antibody titers above the minimum levels required for protection and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias-inducing adjuvant, and protection from virus replication in the lower respiratory tract.IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of nonlive RSV vaccine candidates. The findings presented here underscore the need to evaluate a nonlive RSV vaccine candidate during preclinical development over a wide dose range in the cotton rat RSV enhanced-disease model, as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself and to safe development of nonlive RSV vaccines for seronegative infants and children.

A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease outbreaks in Africa and the Arabian Peninsula. The virus has great potential for transboundary spread due to the presence of competent vectors in non-endemic areas. There is currently no fully licensed vaccine suitable for use in livestock or humans outside endemic areas. Here we report the evaluation of the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. In a previous study, the vaccine elicited strong virus neutralizing antibody responses in sheep and was DIVA (differentiating naturally infected from vaccinated animals) compatible. In the current efficacy study, a group of sheep (n = 5) was vaccinated subcutaneously with the glycoprotein-based subunit vaccine candidate and then subjected to heterologous challenge with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever and absence of RVFV-associated histopathological lesions. We conclude that the subunit vaccine platform represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts.

Isatis indigotica root polysaccharides as adjuvants for an inactivated rabies virus vaccine

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity.

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)–matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes.

Supplementation of dietary germanium biotite enhances induction of the immune responses by foot-and-mouth disease vaccine in cattle.

BackgroundAfter the recent outbreak of foot-and-mouth disease (FMD) in Korea, a vaccination policy has been applied to control the disease. In addition, several non-specific immune stimulators have been used without any scientific evidence that they would enhance the immune response after FMD vaccination and/or protect against FMD. Based on the current situation, the aim of this study was to evaluate the effect of the non-specific immune stimulator germanium biotite on FMD vaccination and immune responses in cattle. To achieve our goal, immune responses to FMD vaccination, such as levels of IgG and IgA, antibody duration, and virus-neutralizing titers were investigated after germanium biotite feeding. The PBMC typing and proliferative response after stimulation with mitogens, the cytokines expression level of PBMC, and the lysozyme activity in the serum were measured to evaluate the immune enhancing effects of germanium biotite following its administration.ResultsFollowing the first vaccination, high level of IgG (at 4 weeks) and IgA (at 2 and 31 weeks) titers in serum and saliva were observed in the germanium biotite-feeding group (p < 0.05). The germanium biotite group also showed high and longstanding inhibition percentage value in ELISA assay at 31 weeks (p < 0.05). Generally, higher virus-neutralizing antibody titers were observed in the feeding group at 20 and 31 weeks after vaccination. Following the feeding germanium biotite, the germanium biotite group showed increased subpopulation of CD4+ lymphocytes and MHC I+II+ cells in PBMCs at 23 week, responding to stimulation of ConA. The levels of IFN-γ (at 3 and 8 weeks), IL-1α (at 3, 11, and 23 weeks), IL-1β (at 3, 8, and 11 weeks), and IL-4 (at 8 and 11 weeks) gene expression were also significantly increased in the feeding group (p < 0.01 and p < 0.05). Feeding with germanium biotite increased the lymphocytes’ proliferative response to the stimulation of ConA and LPS at 23 weeks and lysozyme activity at 9 weeks after feeding.ConclusionsThese results suggest that germanium biotite feeding could increase the protection against FMD virus infection via the induction of higher humoral and cellular immune responses in cattle.

Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice

BackgroundSince its first appearance in the USA in 1999, West Nile virus (WNV) has spread in the Western hemisphere and continues to represent an important public health concern. In the absence of effective treatment, there is a medical need for the development of a safe and efficient vaccine. Live attenuated WNV vaccines have shown promise in preclinical and clinical studies but might carry inherent risks due to the possibility of reversion to more virulent forms. Subunit vaccines based on the large envelope (E) glycoprotein of WNV have therefore been explored as an alternative approach. Although these vaccines were shown to protect from disease in animal models, multiple injections and/or strong adjuvants were required to reach efficacy, underscoring the need for more immunogenic, yet safe DIII-based vaccines.ResultsWe produced a conjugate vaccine against WNV consisting of recombinantly expressed domain III (DIII) of the E glycoprotein chemically cross-linked to virus-like particles derived from the recently discovered bacteriophage AP205. In contrast to isolated DIII protein, which required three administrations to induce detectable antibody titers in mice, high titers of DIII-specific antibodies were induced after a single injection of the conjugate vaccine. These antibodies were able to neutralize the virus in vitro and provided partial protection from a challenge with a lethal dose of WNV. Three injections of the vaccine induced high titers of virus-neutralizing antibodies, and completely protected mice from WNV infection.ConclusionsThe immunogenicity of DIII can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. The superior immunogenicity of the conjugate vaccine with respect to other DIII-based subunit vaccines, its anticipated favourable safety profile and low production costs highlight its potential as an efficacious and cost-effective prophylaxis against WNV.

Expression of MIP-1alpha (CCL3) by a recombinant rabies virus enhances its immunogenicity by inducing innate immunity and recruiting dendritic cells and B cells.

Previously, we showed that overexpression of MIP-1alpha in mouse brain further decreased rabies virus (RABV) pathogenicity (L. Zhao, H. Toriumi, Y. Kuang, H. Chen, and Z. F. Fu, J. Virol., 83:11808-11818, 2009). In the present study, the immunogenicity of recombinant RABV expressing MIP-1alpha (rHEP-MIP1alpha) was determined. It was found that intramuscular immunization of BALB/c mice with rHEP-MIP1alpha resulted in a higher level of expression of MIP-1alpha at the site of inoculation, increased recruitment of dendritic cells (DCs) and mature B cells into the draining lymph nodes and the peripheral blood, and higher virus-neutralizing antibody titers than immunization with the parent rHEP and recombinant RABVs expressing RANTES (CCL5) or IP-10 (CXCL10). Our data thus demonstrate that expression of MIP-1alpha not only reduces viral pathogenicity but also enhances immunogenicity by recruiting DCs and B cells to the site of immunization, the lymph nodes, and the blood.

Replication‐Deficient Rabies Virus–Based Vaccines Are Safe and Immunogenic in Mice and Nonhuman Primates

Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, approximately 40,000-70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1-2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene-deficient RV induced a more rapid and efficient anti-RV response than did P gene-deficient RV immunization. Furthermore, the M gene-deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene-deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines.

Vaccination with virus-like particles protects mice from lethal infection of Rift Valley Fever Virus

Rift Valley Fever virus (RVFV) regularly accounts for severe and often lethal outbreaks among livestock and humans in Africa. Safe and effective veterinarian and human vaccines are highly needed. We present evidence that administration of RVF virus-like particles (VLPs) induces protective immunity in mice. In an accompanying paper, (Habjan, M., Penski, N., Wagner, V., Spiegel, M., Överby, A.K., Kochs, G., Huiskonen, J., Weber, F., 2009. Efficient production of Rift Valley fever virus-like particles: the antiviral protein MxA can inhibit primary transcription of Bunyaviruses. Virology 385, 400–408) we report the production of these VLPs in mammalian cells. After three subsequent immunizations with 1 × 10 6 VLPs/dose, high titers of virus-neutralizing antibodies were detected; 11 out of 12 mice were protected from challenge and only 1 out of 12 mice survived infection in the control groups. VLP vaccination efficiently suppressed replication of the challenge virus, whereas in the control animals high RNA levels and increasing antibody titers against the nucleocapsid protein indicated extensive viral replication. Our study demonstrates that the RVF VLPs are highly immunogenic and confer protection against RVFV infection in mice. In the test groups, the vaccinated mice did not exhibit any side effects, and the lack of anti-nucleocapsid protein antibodies serologically distinguished vaccinated animals from experimentally infected animals.

Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein, rVP2H, in insect larvae

This study describes an alternative approach to produce rVP2H protein using insect larvae of the cabbage looper Trichoplusia ni as hosts for the expression of the protein. The chimeric rVP2H protein, having an extra six histidine residues at the C-terminus of the VP2, a structural protein of infectious bursal disease virus (IBDV), is a vaccine candidate for the prevention of infectious bursal disease. The chimeric rVP2H protein was expressed in insect larvae in form of virus-like particles, in which they maintain their native immunogenic properties. The expression level of rVP2H protein in T. ni larvae was estimated to be approximately 0.4 mg/g of larvae or 0.2 mg/larvae. The rVP2H particles have a uniform morphology of dodecahedral structure with a size of 23 nm in diameter, and the particles could be affinity-purified in one step with immobilized metal-ion affinity chromatography (IMAC) from the larvae homogenate. The recovery of rVP2H protein was approximately 55% following IMAC and the protein was obtained with a purity of around 90%. An additional purification step of ammonium sulphate precipitation was added to speed up the process of microfiltration and ultrafiltration of the homogenate prior to IMAC. This step enhanced the final purity of rVP2H protein to 99%, demonstrating that the purification protocol developed herein was a powerful strategy for obtaining highly pure rVP2H protein from insect larvae. The immunogenicity and protective properties of the larvae-derived rVP2H protein were evaluated using a chicken protection assay. When larvae-derived rVP2H protein was intramuscularly injected into specific-pathogen-free chickens (20 μg/bird), high titres of virus-neutralizing antibodies were induced and the chickens were protected from the infection of a very virulent strain of IBDV isolated locally.

Induction of protective cytotoxic T cell responses in the presence of high titers of virus-neutralizing antibodies: implications for passive and active immunization.

The effect of preexistent virus-neutralizing antibodies on the active induction of antiviral T cell responses was studied in two model infections in mice. Against the noncytopathic lymphocytic choriomeningitis virus (LCMV), pretreatment with neutralizing antibodies conferred immediate protection against systemic virus spread and controlled the virus below detectable levels. However, presence of protective antibody serum titers did not impair induction of antiviral cytotoxic T lymphocyte (CTL) responses after infection with 10(2) PFU of LCMV. These CTLs efficiently protected mice independent of antibodies against challenge with LCMV-glycoprotein recombinant vaccinia virus; they also protected against otherwise lethal lymphocytic choriomeningitis caused by intracerebral challenge with LCMV-WE, whereas transfused antibodies alone did not protect, and in some cases even enhanced, lethal lymphocytic choriomeningitis. Against the cytopathic vesicular stomatitis virus (VSV), specific CTLs and Th cells were induced in the presence of high titers of VSV-neutralizing antibodies after infection with 10(6) PFU of VSV, but not at lower virus doses. Taken together, preexistent protective antibody titers controlled infection but did not impair induction of protective T cell immunity. This is particularly relevant for noncytopathic virus infections since both virus-neutralizing antibodies and CTLs are essential for continuous virus control. Therefore, to vaccinate against such viruses parallel or sequential passive and active immunization may be a suitable vaccination strategy to combine advantages of both virus-neutralizing antibodies and CTLs.

Feline immunodeficiency virus vaccination: characterization of the immune correlates of protection.

Whole inactivated virus (WIV) vaccines derived from the FL4 cell line protect cats against challenge with feline immunodeficiency virus (FIV). To investigate the correlates of protective immunity induced by WIV, we established an immunization regimen which protected a proportion of the vaccinates against challenge. A strong correlation was observed between high virus neutralizing antibody titers and protection following challenge. To investigate further the immune mechanisms responsible for immunity, all of the vaccinates were rechallenged 35 weeks following the initial challenge. Results of virus isolation from peripheral blood mononuclear cells indicated that 9 of 10 vaccinates were protected from viremia following the second challenge, suggesting that vaccine-induced immunity to FIV persisted for at least 8 months. However, more stringent analysis for evidence of infection revealed that 5 of 10 vaccinates harbored virus in lymphoid tissues. Unlike the protection observed immediately following vaccination, which correlated positively with virus neutralizing antibody titer, the ability to resist a second challenge with FIV was more closely correlated with the induction of Env-specific cytotoxic T-cell activity. The results indicate that both virus-specific humoral immunity and cellular immunity play a role in the protection induced in cats by WIV immunization but their relative importance may be dependent on the interval between vaccination and exposure to virus.