Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein

Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma-FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma-FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat.     

Immunogenicity of virus-like particles containing modified human immunodeficiency virus envelope proteins

Extensive glycosylation and variable loops of the HIV envelope protein (Env) are reported to shield some neutralizing epitopes. Here, we investigated the immunogenicity of mutated HIV Envs presented in virus-like particles (VLPs). We immunized mice with simian human immunodeficiency virus (SHIV) VLPs containing mutant HIV Env with reduced glycosylation (3G), variable loop-deleted mutations (dV1V2), or combinations of both types of mutations (3G-dV2-1G), and evaluated immune responses. Immune sera from mice that received VLPs with modified HIV Envs (3G or dV1V2) showed higher neutralizing activities against the homologous HIV 89.6 virus as well as heterologous viruses when compared with wild type SHIV VLP-immunized mice. Lymphocytes from immunized mice produced HIV Env-specific cytokines, with the 3G-dV2-1G mutant producing high levels of cytokines. Interestingly, both dendritic cells and B cells were found to interact with VLPs suggesting that VLPs are effective immunogens. Therefore, this study suggests that VLPs containing modified HIV Env have the potential to be developed as candidate vaccines capable of inducing cellular and humoral immune responses including neutralizing activities.     

Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats.

A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge. The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli. This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E. The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant. Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies. Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection. In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic. Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats.     

A recombinant envelope protein vaccine against West Nile virus

West Nile (WN) virus is a flavivirus that first appeared in North America in 1999. Since then, more than 600 human deaths and 22,000 equine infections have been attributed to the virus in the United States. We expressed a truncated form of WN virus envelope (E) protein in Drosophila S2 cells. This soluble recombinant E protein was recognized by antibodies from naturally infected horses, indicating that it contains native epitopes. Mice and horses produced high-titer antibodies when immunized with recombinant E protein combined with aluminum hydroxide. Immunized mice were resistant to challenge with a lethal viral dose. Sera from immunized horses, administered to naive mice, conferred resistance against a lethal WN viral challenge. In addition, sera of immunized horses neutralized West Nile virus in vitro, as demonstrated by plaque reduction assays. This recombinant form of E protein, combined with aluminum hydroxide, is a candidate vaccine that may protect humans and horses against WN virus infections.     

Prime-boost vaccination using DNA and whole inactivated virus vaccines provides limited protection against virulent feline immunodeficiency virus

Protection against feline immunodeficiency virus (FIV) has been achieved using a variety of vaccines notably whole inactivated virus (WIV) and DNA. However protection against more virulent isolates, typical of those encountered in natural infections, has been difficult to achieve. In an attempt to improve protection against virulent FIV(GL8), we combined both DNA and WIV vaccines in a "prime-boost" approach. Thirty cats were divided into four groups receiving vaccinations and one unvaccinated control group. Following viral challenge, two vaccinated animals, one receiving DNA alone and one the prime-boost vaccine remained free of viraemia, whilst all controls became viraemic. Animals vaccinated with WIV showed apparent early enhancement of infection at 2 weeks post challenge (pc) with higher plasma viral RNA loads than control animals or cats immunised with DNA alone. Despite this, animals vaccinated with WIV or DNA alone showed significantly lower proviral loads in peripheral blood mononuclear cells and mesenteric lymph node cells, whilst those receiving the DNA-WIV prime-boost vaccine showed significantly lower proviral loads in PBMC, than control animals, at 35 weeks pc. Therefore both DNA and WIV vaccines conferred limited protection against viral challenge but the combination of WIV and DNA in a prime-boost approach appeared to offer no significant advantage over either vaccine alone.     

A recombinant subunit vaccine formulation protects against lethal Nipah virus challenge in cats

Nipah virus (NiV) and Hendra virus (HeV) are closely related deadly zoonotic paramyxoviruses that have emerged and re-emerged over the last 10 years. In this study, a subunit vaccine formulation containing only recombinant, soluble, attachment glycoprotein from HeV (sG(HeV)) and CpG adjuvant was evaluated as a potential NiV vaccine in the cat model. Different amounts of sG(HeV) were employed and sG-induced immunity was examined. Vaccinated animals demonstrated varying levels of NiV-specific Ig systemically and importantly, all vaccinated cats possessed antigen-specific IgA on the mucosa. Upon oronasal challenge with NiV (50,000TCID50), all vaccinated animals were protected from disease although virus was detected on day 21 post-challenge in one animal. The ability to elicit protective systemic and mucosal immunity in this animal model provides significant progress towards the development of a human subunit vaccine against henipaviruses.     

Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens

West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA–WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA–WNV vaccines in other preclinical models and use them as candidate vaccine in humans.     

Functionally-inactive and immunogenic Tat, Rev and Nef DNA vaccines derived from sub-Saharan subtype C human immunodeficiency virus type 1 consensus sequences

The efficacy of cellular immune responses elicited by HIV vaccines is dependent on their strength, durability and antigenic breadth. The regulatory proteins are abundantly expressed early in the viral life cycle and CTL recognition may bring about early killing of infected cells. We synthesised DNA vaccine constructs that encode consensus HIV-1 subtype C Tat, Rev and Nef proteins. Proteins carrying inactivating mutations were tested for functional activity and highly expressing, inactive Tat, Rev and Nef mutants were identified and their reading frames fused into a TatRevNef cassette. Single- and polygene Tat, Rev and/or Nef constructs were immunogenic in BALB/c mice. These constructs may serve to increase the antigenic breadth for an HIV-1 vaccine that is relevant for sub-Saharan Africa.     

The development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease

Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.     

Neutralising antibody response in domestic cats immunised with a commercial feline immunodeficiency virus (FIV) vaccine

Across human and veterinary medicine, vaccines against only two retroviral infections have been brought to market successfully, the vaccines against feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV). FeLV vaccines have been a global success story, reducing virus prevalence in countries where uptake is high. In contrast, the more recent FIV vaccine was introduced in 2002 and the degree of protection afforded in the field remains to be established. However, given the similarities between FIV and HIV, field studies of FIV vaccine efficacy are likely to advise and inform the development of future approaches to HIV vaccination.Here we assessed the neutralising antibody response induced by FIV vaccination against a panel of FIV isolates, by testing blood samples collected from client-owned vaccinated Australian cats. We examined the molecular and phenotypic properties of 24 envs isolated from one vaccinated cat that we speculated might have become infected following natural exposure to FIV. Cats vaccinated against FIV did not display broadly neutralising antibodies, suggesting that protection may not extend to some virulent recombinant strains of FIV circulating in Australia.     

HBV外膜蛋白2型重组腺相关病毒免疫原性

目的观察乙型肝炎病毒(HBV)外膜大蛋白(LP)基因的2型重组腺相关病毒(rAAV-2-LP)在Balb/c小鼠体内的免疫原性。方法以5×1011的rAAV-2-LP单次肌肉注射接种Balb/c小鼠,RT-PCR扩增肝脏组织中HBV外膜大蛋白基因cDNA;RIA法检测血清中表面抗体(抗-HBs)滴度;51Cr释放分析检测细胞毒性T淋巴细胞(CTL)活性。结果HBV外膜大蛋白基因已整合到肝细胞染色体DNA,rAAV-LP接种小鼠后第20 d,小鼠体内可出现针对HBV外膜大蛋白特异性CTL,第30 d,血清中可检测到表面抗体。结论rAAV可以介导HBV外膜大蛋白基因转移到小鼠肝细胞;rAAV-2-LP免疫小鼠可以同时诱导体液和细胞免疫反应的出现。基于rAAV-2载体的HBV疫苗,对于防止HBV感染,尤其是作为慢性乙型肝炎的治疗性疫苗可能具有潜在的应用价值。     

寨卡病毒包膜蛋白重组人腺病毒5型载体的构建与免疫原性测定

目的 以腺病毒5型载体（Ad5）构建含寨卡病毒（ZIKV）包膜蛋白（prM-E）的复制缺陷型5型重组腺病毒（rAd5）表达载体,测定prM-E在细胞中的表达及对小鼠的免疫原性。方法 从ZIKV毒株Z16006（亚洲型）分离获得prM-E基因片段,以重组腺病毒AdMax^TM系统包装重组腺病毒rAd5/prM-E。选用C57BL/6小鼠,以rAd5/prM-E 10⁷、10⁸和10⁹ PFU三个剂量肌内注射免疫小鼠,在第3周同等剂量加强免疫一次,第5周从小鼠眼球取血并分离脾淋巴细胞。分别以ELISpot和ELISA方法测定小鼠对ZIKV prM-E的体液与细胞免疫反应。结果 成功构建复制缺陷型重组腺病毒载体rAd5/prM-E,采用Western blot方法和抗ZIKV E抗体检测rAd5/prM-E感染的293A细胞表达产物,可见与E蛋白相应的56 kDa蛋白带。以ELISpot方法测定小鼠脾淋巴细胞特异性IFN-γ分泌细胞形成斑点数（SFCs）,结果分别为（688.54±186.43）、（1 084.90±144.14）和（1 640.20±147.13） SFCs/10⁶脾细胞,与病毒接种剂量呈正比关系;采用ELISA测定免疫小鼠血清中抗E抗体,其滴度（log10值）分别为（3.14±0.39）、（3.50±0.30）和（3.74±0.25）, 均高于对照组小鼠（0.80±0.17）,差异具有统计学意义（P<0.001）。结论 rAd5/prM-E具有感染小鼠并诱导小鼠产生强烈的特异性抗体和细胞免疫反应的能力,表明prM-E具有良好的免疫原性,为ZIKV候选疫苗研制提供了可靠的免疫源。     

Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein

Recombinant plasmid DNA constructs expressing truncated or full-length dengue-1 envelope (E) with or without the pre-membrane (prM) were tested for immunogenicity in mice, as candidate dengue DNA vaccines. Two plasmids, one expressing the N-terminal 80% E and the other expressing prM and full length E were immunogenic in intradermally inoculated mice. The vaccinated mice produced dengue-1 specific antibodies that were both neutralizing and long lasting. Data suggested that the plasmid expressing prM and full length E produced virus like particles in transfected cells, and is probably a better immunogen compared to that expressing 80% E.     

Evaluation of recombinant E2 protein-based and whole-virus inactivated candidate vaccines against chikungunya virus

Objectives

With the re-emergence of chikungunya virus (CHIKV) in an explosive form and in the absence of a commercially available vaccine, we aimed to develop candidate vaccines employing recombinant E2 protein or chemically inactivated whole virus.

Design and methods

E2 gene of CHIKV isolate of ECSA genotype was cloned in pET15b vector, expressed and purified (rE2p). The virus was propagated in Vero cell line, purified and inactivated with formalin and BPL individually. Six to eight weeks old female BALB/c mice were immunized intramuscularly with two doses of 10 μg, 20 μg and 50 μg of vaccine formulations with or without adjuvants, 2 weeks apart. The adjuvants evaluated were alum, Mw, CadB (rE2p), alum/Mw (formalin inactivated CHIKV) and alum (BPL-inactivated CHIKV). Humoral immunity was assessed by ELISA and in vitro neutralization test using homologous and heterologous (Asian genotype) strains of CHIKV. Two cohorts of vaccinated mice were challenged separately via intranasal route with homologous virus two and 20 weeks after the 2nd dose. Viral load (CHIKV RNA by real time PCR) was determined in the serum and tissues (muscle, brain, spleen) of the mice challenged with the homologous virus.

Results

Anti-CHIK-antibody titres were dose dependent for all the immunogen formulations. BPL-inactivated vaccines led to the highest ELISA/neutralizing antibody (nAb) titres while alum was the most effective adjuvant. Asian genotype strain could be neutralized by the nAbs. In an adult mouse model, complete protection was offered by the alum-adjuvanted rE2p and both the inactivated vaccines as no virus was detected in the tissues and blood after challenge 2 weeks or 20 weeks-post-2nd dose. However, with rE2p-CadB, very low viremia was recorded on the 2nd day-post-challenge.

Conclusion

Both rE2p and BPL/formalin-inactivated virus are promising candidate vaccines deserving further evaluation.     

Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity

Zika virus (ZIKV) reemergence poses a significant health threat especially due to its risks to fetal development, necessitating safe and effective vaccines that can protect pregnant women. Zika envelope domain III (ZE3) has been identified as a safe and effective vaccine candidate, however it is poorly immunogenic. We previously showed that plant-made recombinant immune complex (RIC) vaccines are a robust platform to improve the immunogenicity of weak antigens. In this study, we altered the antigen fusion site on the RIC platform to accommodate N-terminal fusion to the IgG heavy chain (N-RIC), and thus a wider range of antigens, with a resulting 40% improvement in RIC expression over the normal C-terminal fusion (C-RIC). Both types of RICs containing ZE3 were efficiently assembled in plants and purified to >95% homogeneity with a simple one-step purification. Both ZE3 RICs strongly bound complement receptor C1q and elicited strong ZE3-specific antibody titers that correlated with ZIKV neutralization. When either N-RIC or C-RIC was codelivered with plant-produced hepatitis B core (HBc) virus-like particles (VLP) displaying ZE3, the combination elicited 5-fold greater antibody titers (>1,000,000) and more strongly neutralized ZIKV than either RICs or VLPs alone, after only two doses without adjuvant. These findings demonstrate that antigens that require a free N-terminus for optimal antigen display can now be used with the RIC system, and that plant-made RICs and VLPs are highly effective vaccines targeting ZE3. Thus, the RIC platform can be more generally applied to a wider variety of antigens.     

Evaluation of human immunodeficiency virus type 1 subtype C gag, pol, and gagpol DNA and alphavirus replicon vaccines

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8(+) T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press) and support a subtype C in-frame gagpol fusion gene vaccine.     

A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in nonhuman primates

We have previously reported the construction and evaluation in mice of recombinant fusion proteins formed by a fragment (aa 286-426) of the dengue envelope protein and the P64k protein from Neisseria meningitidis. In this work we describe the immunization of Macaca fascicularis monkeys with two variants of these proteins [PD3 (insertion variant) and PD5 (fusion variant)] corresponding to serotype 2. Four doses of the proteins adjuvated in Freund's adjuvant were administered and the kinetics of antibody induction was monitored by ELISA and neutralization tests. Monkeys receiving PD3 or PD5 developed functional antibodies (Abs) in a dose-dependent manner. Following challenge with 5 log PFU of wild type dengue-2 virus (DEN2), animals immunized with PD5 were protected from developing viremia. These results constitute a proof-of-concept demonstrating that a fragment of the dengue envelope protein, containing the domain III and produced as a recombinant fusion protein in Escherichia coli, induces functional and protective immunity in a nonhuman primate model.     

Evaluation of safety and immunogenicity of feline vaccines with reduced volume

A non adjuvanted vaccine against feline herpesvirus, feline calicivirus, feline panleucopenia and feline leukemia has been formulated in reduced volume (0.5 ml) with the same antigen content as the conventional 1 ml presentation. This paper reports studies evaluating the safety and the immunogenicity of this reduced volume vaccine in comparison with the conventional volume vaccine. The safety of both vaccines was evaluated in a small sized laboratory trial. It was further tested in a randomized controlled field trial on a total of 398 cats. Immediate and delayed local and systemic adverse events were monitored after vaccination. The immunogenicity of each vaccine was also checked by serological antibody responses against the vaccines antigens during the laboratory trial.These studies showed that the 0.5 ml vaccine was well tolerated in cats, inducing less local events, while keeping the same immunogenicity as the corresponding 1 ml vaccine. Reducing the volume of the vaccine is a way to improve the convenience of administration and to help following vaccination guidelines with the aim of reducing the incidence of adverse events following vaccination.     

Immunogenicity and efficacy of recombinant subunit vaccines against phocid herpesvirus type 1

Phocid herpesvirus type 1 (PhHV-1) is an alpha-herpesvirus that causes significant morbidity and mortality among young and immunocompromised harbour seals (Phoca vitulina) and therefore represents a major problem for seal rehabilitation centres. Consequently, there is a need for a safe and effective PhHV-1 vaccine. We tested an ISCOM-based recombinant PhHV-1 gB vaccine alone (gB) or with the addition of recombinant PhHV-1 gD (gBD) for (i). immunogenicity and protective efficacy against feline herpesvirus (FHV) infection in cats and (ii). their immunogenicity in seals. The FHV-cat model was chosen based on the close antigenic relationship between PhHV-1 and FHV. Upon challenge, all vaccinated (gB and gBD) cats excreted significantly less FHV (P<0.01) and gBD vaccinated cats showed less weight loss (P=0.05) than the mock-vaccinated cats. However, adding gD to the gB vaccine did not result in significantly better protection. Based on these data, immunogenicity studies in seals under rehabilitation were performed with the gB vaccine only. To this end, gB vaccine was tested at two different doses (20 or 40 microg). PhHV-1 specific antibody titres and in vitro proliferative T cell responses were measured in all seals upon vaccination. No differences were observed in antibody titres between seals vaccinated with either 20 or 40 microgB, but the higher gB concentration did result in higher specific proliferative T cell responses (P<0.01). Based on the close antigenic relationship between PhHV-1 and FHV, the safety and efficacy data in the FHV-cat model, and the immunogenicity data in the vaccinated seals, the gB based vaccine is expected to be safe and effective in protecting against PhHV-1 related disease in harbour seals.