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The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans 1 . CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 μg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates., (© 2021. The Author(s).)

Only fifteen months after the beginning of the COVID-19 pandemic, several vaccines are already available for clinical use. While the spike protein of SARS-CoV-2 constitutes the main target of all predominant SARS-CoV-2 vaccines, they work by different mechanisms (mRNA-based vaccines vs. vector-based vaccines vs. protein-based vaccines).Though there are slight differences regarding the level of protection against mild COVID-19, all five vaccines that have been through phase 3 trials were nearly 100 % effective in preventing severe or fatal cases of COVID-19. The side effects were of short duration.Patients with chronic kidney disease (or other significant comorbidities) were largely excluded from Phase 3 trials, which makes definite recommendations concerning their vaccination difficult. The vaccine's effectiveness may be reduced in that population due to a uremic immune defect and/or immunosuppressive medication. However, these patients have an increased risk for severe or fatal COVID-19, so that they may particularly benefit from the vaccine., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD's Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.

The H9N2 avian influenza virus has been widely spread in poultry around the world. It is proved to the world that the avian influenza virus can directly infect human beings without any intermediate host adaptation in "1997 Hong Kong avian influenza case," which shows that the avian influenza virus not only causes significant losses to the poultry industry but also affects human health. In this study, we aimed to address the problem of low protection of avian H9N2 subtype influenza virus vaccine against H9N2 wild-type virus. We have rescued the H9.4.2.5 branched avian influenza virus isolated in South China by reverse genetics technology. We have recombined these virus (rHA/NA-GD37 and rHA/NA-GD38) which contain hemagglutinin and neuraminidase genes from the H9N2 avian influenza virus (MN064850 or MN064851) and 6 internal genes from the avian influenza virus (KY785906). We compared the biological properties of the virus for example virus proliferation, virus elution, thermostability, and pH stability. Then, we evaluated the immune effects between rHA/NA-GD37 and GD37, which show that the recombinant avian influenza virus-inactivated vaccine can stimulate chickens to produce higher antibody titers and produce little inflammatory response after the challenge. It is noticeable that the recombinant virus-inactivated vaccine had better immune impact than the wild-type inactivated vaccine. Generally speaking, this study provides a new virus strain for the development of a H9N2 vaccine., (Copyright © 2020. Published by Elsevier Inc.)

Nipah virus (NiV) is a re-emerging zoonotic pathogen that causes high mortality in humans and pigs. Oral immunization in free-roaming animals is one of the most practical approaches to prevent NiV pandemics. We previously generated a recombinant rabies viruses (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, rERAG 333E , which contains a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G 333E ) and serves as an oral vaccine for dog rabies. In this study, we generated two recombinant RABVs, rERAG 333E /NiVG and rERAG 333E /NiVF, expressing the NiV Malaysian strain attachment glycoprotein (NiV-G) or fusion glycoprotein (NiV-F) gene based on the rERAG 333E vector platform. Both rERAG 333E /NiVG and rERAG 333E /NiVF displayed growth properties similar to those of rERAG 333E and caused marked syncytia formation after co-infection in BSR cell culture. Adult and suckling mice intracerebrally inoculated with the recombinant RABVs showed NiV-G and NiV-F expression did not increase the virulence of rERAG 333E . Oral vaccination with rERAG 333E /NiVG either singularly or combined with rERAG 333E /NiVF induced significant NiV neutralizing antibody against NiV and RABV, and IgG to NiV-G or NiV-F in mice and pigs. rERAG 333E /NiVG and rERAG 333E /NiVF thus appeared to be suitable candidates for further oral vaccines for potential animal targets in endemic areas of NiV disease and rabies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Recombinant viral vaccines expressing antigens of pathogenic microbes (e.g., HIV, Ebola virus, and malaria) have been designed to overcome the insufficient immune responses induced by the conventional vaccines. Our knowledge of and clinical experience with the new recombinant viral vaccines are insufficient, and a clear regulatory pathway is needed for the further development and evaluation of recombinant viral vaccines. In 2018, the research group supported by the Ministry of Health, Labour and Welfare, Japan (MHLW) published a concept paper to address the development of recombinant viral vaccines against infectious diseases. Herein we summarize the concept paper-which explains the Japanese regulatory concerns about recombinant viral vaccines-and provide a focus of discussion about the development of recombinant viral vaccines., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The 2014 Ebola outbreak in West Africa brought great threat to public health worldwide. There was no approved antiviral therapy or vaccine available to control the disease at that time. Several kinds of Ebola vaccines were urgently under development across the world. Among these, the novel recombinant adenovirus type 5 vector-based Ebola vaccine (Ad5-EBOV)-the first Ebola vaccine based on the 2014 Zaire Guinea epidemic strain-was developed in China, and its safety and immunogenicity were demonstrated in China and Sierra Leone. The license to market the drug was approved on October 19, 2017, by the Chinese Food and Drug Administration. In order to standardize the test on the Ad5-EBOV virus titer, China's national standard substance for the virus titer of Ad5-EBOV was established according to the recommendations for the preparation, characterization, and establishment of international and other biological reference standards from the World Health Organization and Chinese Pharmacopoeia (third edition). The standard for the Ad5-EBOV virus titer was prepared with a volume of 0.5 mL per ampoule in lyophilized form. The samples of the standard, designated as A, B, C, D with different aims, were blinded and distributed to five laboratories to be collaboratively calibrated. The virus titer for this standard was determined with the antibody staining method according to the instructions in the Adeno-X™ Rapid Titer Kit. The homogeneity and stability of the standard substance were also satisfied. The virus titer standard value was 8.54 lg infectious units (IFU)/mL, and the 95% confidence interval was between 7.94 lg IFU/mL and 9.14 lg IFU/mL. This standard was approved by the Chinese national committee and is available on the National Institutes for Food and Drug Control Web site ( www.nifdc.org.cn ; lot no. 250019-201501).

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The risk of developing herpes zoster (HZ) increases with age and is thought to be associated with a decrease in cell-mediated immunity in older adults. The adjuvanted varicella-zoster virus (VZV) glycoprotein E (gE) recombinant subunit vaccine (HZ/su) showed >90% efficacy in the prevention of HZ when administered in adults ≥50 years of age. Here we aim to evaluate immunogenicity consistency of 3 different HZ/su vaccine lots and to assess safety of these lots., Methods: This multicenter, phase III, double-blind, randomized study (NCT02075515), assessed lot-to-lot consistency in terms of immunogenicity of HZ/su and also assessed safety of these lots. Participants aged 50 years or older were randomized (1:1:1) to receive 2 doses of HZ/su, 2 months apart, from 1 out of 3 randomized HZ/su lots (Lots A, B and C). Humoral immunogenicity was assessed pre-vaccination and 1 month post-second vaccination by anti-gE antibody enzyme-linked immunosorbent assay. Lot-to-lot consistency was demonstrated if the 2-sided 95% confidence intervals of the anti-gE geometric mean concentration ratio between all lot pairs were within 0.67 and 1.5. Solicited symptoms were recorded within 7 days and unsolicited adverse events (AEs) within 30 days after each vaccination. Serious AEs (SAEs) and potential immune-mediated diseases (pIMDs) were reported until study end (12 months post-second vaccination)., Results: Of 651 participants enrolled in the study, 638 received both doses of the HZ/su vaccine and 634 completed the study. Humoral immune responses were robust and consistency between 3 manufacturing lots was demonstrated. The incidence of solicited symptoms, unsolicited AEs and SAEs was comparable between all lots. Three fatal SAEs, 1 in each lot, were reported, none of which were considered vaccine-related by investigator assessment. Two out of the 8 reported pIMDs were considered vaccine-related by the investigator., Conclusion: The three HZ/su manufacturing lots demonstrated consistent immunogenicity. No safety concerns were identified. Clinical trial registry number: NCT02075515 (ClinicalTrials.gov)., (Copyright © 2017 GlaxoSmithKline SA. Published by Elsevier Ltd.. All rights reserved.)

Background: Large Phase III trials across Asia and Latin America have recently demonstrated the efficacy of a recombinant, live-attenuated dengue vaccine (Dengvaxia) over the first 25 mo following vaccination. Subsequent data collected in the longer-term follow-up phase, however, have raised concerns about a potential increase in hospitalization risk of subsequent dengue infections, in particular among young, dengue-naïve vaccinees. We here report predictions from eight independent modelling groups on the long-term safety, public health impact, and cost-effectiveness of routine vaccination with Dengvaxia in a range of transmission settings, as characterised by seroprevalence levels among 9-y-olds (SP9). These predictions were conducted for the World Health Organization to inform their recommendations on optimal use of this vaccine., Methods and Findings: The models adopted, with small variations, a parsimonious vaccine mode of action that was able to reproduce quantitative features of the observed trial data. The adopted mode of action assumed that vaccination, similarly to natural infection, induces transient, heterologous protection and, further, establishes a long-lasting immunogenic memory, which determines disease severity of subsequent infections. The default vaccination policy considered was routine vaccination of 9-y-old children in a three-dose schedule at 80% coverage. The outcomes examined were the impact of vaccination on infections, symptomatic dengue, hospitalised dengue, deaths, and cost-effectiveness over a 30-y postvaccination period. Case definitions were chosen in accordance with the Phase III trials. All models predicted that in settings with moderate to high dengue endemicity (SP9 ≥ 50%), the default vaccination policy would reduce the burden of dengue disease for the population by 6%-25% (all simulations: -3%-34%) and in high-transmission settings (SP9 ≥ 70%) by 13%-25% (all simulations: 10%- 34%). These endemicity levels are representative of the participating sites in both Phase III trials. In contrast, in settings with low transmission intensity (SP9 ≤ 30%), the models predicted that vaccination could lead to a substantial increase in hospitalisation because of dengue. Modelling reduced vaccine coverage or the addition of catch-up campaigns showed that the impact of vaccination scaled approximately linearly with the number of people vaccinated. In assessing the optimal age of vaccination, we found that targeting older children could increase the net benefit of vaccination in settings with moderate transmission intensity (SP9 = 50%). Overall, vaccination was predicted to be potentially cost-effective in most endemic settings if priced competitively. The results are based on the assumption that the vaccine acts similarly to natural infection. This assumption is consistent with the available trial results but cannot be directly validated in the absence of additional data. Furthermore, uncertainties remain regarding the level of protection provided against disease versus infection and the rate at which vaccine-induced protection declines., Conclusions: Dengvaxia has the potential to reduce the burden of dengue disease in areas of moderate to high dengue endemicity. However, the potential risks of vaccination in areas with limited exposure to dengue as well as the local costs and benefits of routine vaccination are important considerations for the inclusion of Dengvaxia into existing immunisation programmes. These results were important inputs into WHO global policy for use of this licensed dengue vaccine., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: LC is employed by Sanofi Pasteur. KV is a staff member of the World Health Organization. TAP and GE receive support from GlaxoSmithKline for unrelated work on dengue vaccine modelling. IL, TJH, and CABP have received travel support from Sanofi Pasteur to present other work on dengue vaccine modelling. Sanofi Pasteur has not funded any of their research and was not involved in any research decisions related to their work presented. NF gave advice to Sanofi-Pasteur and the World Health Organization on the efficacy profile and potential public health impact of Dengvaxia. He is also collaborating with Sanofi-Pasteur on secondary analyses of Dengvaxia clinical trial data. He has received no remuneration, grant income, expense payments or in-kind benefit from Sanofi-Pasteur. DATC and IRB have advised WHO on the use of the Sanofi vaccine in a number of meetings and as part of a consortium of modelers who estimated the potential impact of the vaccine. On occasion they received travel expenses for visits to WHO. They have also advised Sanofi Pasteur Ltd. on the implications their work has for use of their vaccine. They have not received any financial or in-kind payment from Sanofi. All other authors have declared that no competing interests exist.

Hepatitis E virus infections have been continuously reported in Indian subcontinent, Africa, southeast and central Asia, posing great health threats to the public, especially to pregnant women. Hecolin® is the only licensed HEV vaccine developed by Xiamen Innovax Biotech Co., Ltd. Extensive characterizations on antigenicity, physicochemical properties, efficacy in clinical trials, and manufacturing capability have made Hecolin® a promising vaccine for HEV control. However, there are many obstacles in large scale application of Hecolin®. Efforts are needed to further evaluate safety and efficacy in HEV risk populations, and to complement HEV standards for quality control. Passing World Health Organization prequalification and licensing outside China are priorities as these are also hindering Hecolin® promotion. Multilateral cooperation among Chinese vaccine manufacturers, Chinese National Regulatory Authorization (NRA) and WHO will expedite the entrance of Hecolin® into international market, so that Hecolin® could play its due role in global hepatitis E control.

The efficacy of commercially available recombinant herpesvirus of turkeys-infectious bursal disease (rHVT-IBD) virus vaccines was studied in broiler chickens derived from an IBDV-vaccinated breeder flock at 30 wk of age (Trial 1) and 60 wk of age (Trial 2). In parallel, specific-pathogen-free (SPF) white leghorn chickens were used to evaluate vaccine efficacy to control for the effects of maternally derived antibodies (MDA) associated with the broiler chickens. Broilers and SPF leghorns were vaccinated subcutaneously in the neck at 1 day of age with Vaxxitek® HVT+IBD or Vectormune® HVT-IBD vaccines and were placed in isolators. On 10, 14, 18, 22, and 26 days postvaccination (DPV), vaccinated and nonvaccinated broilers and SPF leghorns were bled prior to challenge via the oral-nasal route with infectious bursal disease (IBD) reference strains ST-C, Delaware variant E (Del E), or contemporary field isolates DMV/5038/07 or FF6. Microscopic lesion assessment of the bursa was useful for assessing IBDV challenge in both rHVT-IBD-vaccinated broiler and SPF leghorn chickens. In general, rHVT-IBD vaccines induced greater protection as the time between vaccination and challenge increased. Based on incidence of microscopic lesions (IML) of bursa tissue, Vaxxitek HVT+IBD vaccination of SPF leghorns induced protection by 18 DPV and continued to protect 22 DPV and 26 DPV in Trials 1 and 2. Vectormune HVT-IBD vaccine induced protection of SPF leghorns by 18 or 22 DPV in Trial 1, depending upon the IBDV challenge strain. However, the onset of protection was delayed until 22 or 26 DPV in Trial 2. With either commercial vaccine, rHVT-IBD vaccination of broiler chickens was not as effective as was observed in SPF leghorns, based on IML of bursa tissue. However, Vaxxitek HVT+IBD vaccination protected broilers following challenge with ST-C in both Trial 1 (30-wk-old breeder progeny) and Trial 2 (60-wk-old breeder progeny). Partial protection against FF6 (Trial 1) and DMV/5038/07 (Trial 2) challenges was observed. Vectormune HVT-IBD vaccination protected broilers vs. FF6 challenge in Trial 1. In Trial 2, the vaccine did not offer protection on the basis of IML of bursa tissue. The results indicate that 1) bursa/body weight ratios were not consistently useful as a tool for assessing IBDV challenge in broiler chickens with anti-IBDV MDA compared to assessment by IML of bursa tissue, though were useful for assessing protection in SPF leghorns; and 2) both vaccines may offer some protection to older broilers; however, a window of susceptibility exists between the waning of MDA and the development of vaccine-induced antibodies. The SPF studies showed that some vaccinated chickens were not protected from an IBDV challenge earlier than 14 DPV while broiler studies showed that MDA was not fully protective beyond 10 DPV. Because these vaccines did not protect chickens from an IBDV challenge during this window of susceptibility, our data show that breeder vaccination programs for IBDV must aim to maximize anti-IBDV MDA in progeny to protect against early IBDV challenge.

The increasing diversity of influenza strains circulating in swine herds escalates the potential for the emergence of novel pandemic viruses and highlights the need for swift development of new vaccines. Baculovirus has proven to be a flexible platform for the generation of recombinant forms of hemagglutinin (HA) including subunit, VLP-displayed, and baculovirus-displayed antigens. These presentations have been shown to be efficacious in mouse, chicken, and ferret models but little is known about their immunogenicity in pigs. To assess the utility of these HA presentations in swine, Baculovirus constructs expressing HA fused to swine IgG2a Fc, displayed in a FeLV gag VLP, or displayed in the baculoviral envelope were generated. Vaccines formulated with these antigens wer The e administered to groups of pigs who were subsequently challenged with H1α cluster H1N1 swine influenza virus (SIV) A/Swine/Indiana/1726/88. Our results demonstrate that vaccination with any of these three vaccines elicits robust hemagglutinin inhibition titers in the serum and decreased the severity of SIV-associated lung lesions after challenge when compared to placebo-vaccinated controls. In addition, the number of pigs with virus detected in the lungs and nasal passages was reduced. Taken together, the results demonstrate that these recombinant approaches expressed with the baculovirus expression vector system may be viable options for development of SIV vaccines for swine., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tick infestation may cause several problems including affecting domestic animal health and reducing the production of meat and milk, among others. Resistance to several classes of acaricides have been reported, forcing researchers to search for alternative measures, such as vaccines against ticks, to ensure tick control while having no or at least low negative impacts on the environment and public health. However, the current commercially available vaccines in different strains of Rhipicephalus microplus are reported to be of low efficacy. Fortunately, reverse vaccinology approaches have shown positive results in the new generation of vaccines. On this basis, a synthetic peptide from the ATAQ protein, which is present in the gut and Malpighi tubes of R. microplus, was synthesized. The ATAQ proteins were isolated, characterized and sequenced from several species of the genus Rhipicephalus. The alignment showed 93.3% identity among DNA sequences of ATAQs from these species. Because of this, immunization trials with this peptide were conducted on mice, rabbits and cattle to evaluate the humoral immune response and the efficacy against Rhipicephalus sanguineus in addition to R. microplus. Based on recent results, we conclude that reverse vaccinology is a promising approach because it is more accurate and faster than conventional methods in the detection of potential antigens to use in anti-tick vaccines. It is not only applicable against R. microplus but also against tick species that play important roles in spreading other diseases. ATAQ proteins should be considered as the antigen in new trials to develop a multi-antigenic vaccine. Although these peptides behave as hapten and are not able to be recognized by the immune system on its own, using carriers and adjuvants helps its presentation and induces strong immune responses. Furthermore, an efficiency of 35% reduction in overall life cycle parameters was reported for R. microplus (98% for ELISA responder animals) and 47% for R. sanguineus. Although not yet enough to prevent the environment to infestation of ticks, this still constitutes a promising strategy that could be applied to integrated measures on tick control and in new research that develops anti-tick vaccines., (Copyright © 2016 Elsevier B.V. All rights reserved.)

There have been only a few antigen genes of Eimeria brunetti reported up to now. In this study, the gene encoding the microneme protein 2 (EbMIC2) was isolated from oocysts of E. brunetti by RT-PCR and the immunogenicity of recombinant EbMIC2 was observed. The EbMIC2 was cloned into vector pMD19-T for sequencing. The sequence was compared with the published EbMIC2 gene from GenBank revealed homology of the nucleotide sequence and amino acids sequence were 99.43 and 98.63%, respectively. The correct recombinant pMD-EbMIC2 plasmid was inserted into the pET-28a (+) expressing vector and transformed into competent Escherichia coli BL21 cells for expression. The expressed product was analyzed using SDS-PAGE and Western-blot. The results indicated that the recombinant EbMIC2 protein was recognized strongly by serum from naturally infected chicken with E. brunetti. Rat rcEbMIC2 antisera bound to bands of about 36 kDa in the somatic extract of E. brunetti sporozoites. The recombinant plasmid pVAX1-EbMIC2 was constructed and then the efficacies of recombinant plasmid and recombinant protein were evaluated. The results of IgG antibody level and cytokines concentration suggested that recombinant EbMIC2 could increase the IgG antibody level and induce the expressions of cytokines. Animal challenge experiments demonstrated that the recombinant EbMIC2 protein and recombinant plasmid pVAX1-EbMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented high anti-coccidial index. All results suggested that EbMIC2 could become an effective candidate for the development of new vaccine against E. brunetti infection., (Published by Elsevier Inc.)

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Poxviruses as expression vectors are widely used in medical research for the development of recombinant vaccines and molecular therapies. Here we review recent accomplishments in vaccine research using recombinant modified vaccinia virus ankara (MVA). MVA is a highly attenuated vaccinia virus strain that originated from serial tissue culture passage in chicken embryo fibroblasts more than 40 years ago. Growth adaptation to avian host cells caused deletions and mutations in the viral genome affecting about 15% of the original genetic information. In consequence, MVA is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional vaccinia virus. Because of its safety for the general environment MVA can be handled under conditions of biosafety level one. Non-replicating MVA can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. Therefore, recombinant MVA have been established as an extremely safe and efficient vector system for vaccine development in medical research. By now, various recombinant MVA vaccines have been found safe and immunogenic when used for phase I/II clinical testing in humans, and suitable for industrial scale production following good practice of manufacturing. Thus, there is an obvious usefulness of recombinant MVA vaccines for novel prophylactic and therapeutic approaches also in veterinary medicine. Results from first studies in companion and farm animals are highly promising.

Chimeric DNA vaccines encoding Eimeria tenella (E. tenella) surface antigen 5401 were constructed and their efficacies against E. tenella challenge were studied. The open reading frame (ORF) of 5401 was cloned into the prokaryotic expression vector pGEX-4T2 to express the recombinant protein and the expressed recombinant protein was identified by Western blot. The ORF of 5401 and chicken cytokine gene IFN-γ or IL-2 were cloned into the eukaryotic expression vector pVAX1 consecutively to construct DNA vaccines pVAX-5401-IFN-γ, pVAX-5401-IL-2 and pVAX-5401. The expression of aim genes in vivo was detected by reverse transcription-polymerase chain reaction and Western blot. Fourteen-day-old chickens were inoculated twice at an interval of 7 days with 100 µg of plasmids pVAX-5401, pVAX-5401-IFN-γ and pVAX-5401-IL-2 or 200 µg of recombinant 5401 protein by leg intramuscular injection, respectively. Seven days after the second inoculation, all chickens except the unchallenged control group were challenged orally with 5 × 10(4) sporulated oocysts of E. tenella. Seven days after challenge, all chickens were weighted and slaughtered to determine the effects of immunization. The results showed the recombinant protein was about 90 kDa and reacted with antiserum against soluble sporozoites. The animal experiment showed that all the DNA vaccines pVAX-5401, pVAX-5401-IFN-γ or pVAX-5401-IL-2 and the recombinant 5401 protein could obviously alleviate body weight loss and cecal lesions as compared with non-vaccinated challenged control and empty vector pVAX1control. Furthermore, pVAX-5401-IFN-γ or pVAX-5401-IL-2 induced anti-coccidial index (ACI) of 180.01 or 177.24 which were significantly higher than that of pVAX-5401. The results suggested that 5401 was an effective candidate antigen for vaccine. This finding also suggested that chicken IFN-γ or IL-2 could effectively improve the efficacies of DNA vaccines against avian coccidiosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P<0.001). Levels of antibodies against rNcGRA7 were statistical higher in group A animals when compared with groups B and C at weeks 5 and 16 (P>0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or inactivated whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 lysate vaccine adjuvanted with Iscom Matrix to generate a longer lasting specific antibody response in blood and milk, with improved opsonic capacity, compared with a S. aureus CP5 whole-cell formulation. The aim of the present study was to obtain an experimental immunogen composed of lysed cells of a CP5 S. aureus strain supplemented with recombinant clumping factor A, fibronectin binding protein A and β-toxin formulated with Iscom Matrix, characterize the immune response generated when immunizing pregnant heifers and assess the functional role of antibodies raised against this immunogen in experimental models. Both a lysate vaccine and a lysate+recombinant antigens vaccine elicited antibodies that promoted neutrophil phagocytosis and inhibited internalization into mammary epithelial cells, in vitro. Incorporation of defined antigenic molecules to the lysate formulation elicited a strong specific humoral immune response against both lysate and recombinant antigens and was associated with higher expression of regulatory and pro-inflammatory cytokines. In addition, antibodies were efficient for blocking S. aureus binding to bovine fibrinogen and fibronectin, and neutralizing β-toxin effect in vitro, placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: For regulatory approval, consistency in manufacturing of vaccine lots is expected to be demonstrated in confirmatory immunogenicity studies using two-sided equivalence trials. This randomized, double-blind study (NCT01323972) assessed consistency of three RTS,S/AS01 malaria vaccine batches formulated from commercial-scale purified antigen bulk lots in terms of anti-CS-responses induced., Methods: Healthy children aged 5-17 months were randomized (1:1:1:1) to receive RTS,S/AS01 at 0-1-2 months from one of three commercial-scale purified antigen bulk lots (1600 litres-fermentation scale; commercial-scale lots), or a comparator vaccine batch made from pilot-scale purified antigen bulk lot (20 litres-fermentation scale; pilot-scale lot). The co-primary objectives were to first demonstrate consistency of antibody responses against circumsporozoite (CS) protein at one month post-dose 3 for the three commercial-scale lots and second demonstrate non-inferiority of anti-CS antibody responses at one month post-dose 3 for the commercial-scale lots compared to the pilot-scale lot. Safety and reactogenicity were evaluated as secondary endpoints., Results: One month post-dose-3, anti-CS antibody geometric mean titres (GMT) for the 3 commercial scale lots were 319.6 EU/ml (95% confidence interval (CI): 268.9-379.8), 241.4 EU/ml (207.6-280.7), and 302.3 EU/ml (259.4-352.3). Consistency for the RTS,S/AS01 commercial-scale lots was demonstrated as the two-sided 95% CI of the anti-CS antibody GMT ratio between each pair of lots was within the range of 0.5-2.0. GMT of the pooled commercial-scale lots (285.8 EU/ml (260.7-313.3)) was non-inferior to the pilot-scale lot (271.7 EU/ml (228.5-323.1)). Each RTS,S/AS01 lot had an acceptable tolerability profile, with infrequent reports of grade 3 solicited symptoms. No safety signals were identified and no serious adverse events were considered related to vaccination., Conclusions: RTS,S/AS01 lots formulated from commercial-scale purified antigen bulk batches induced a consistent anti-CS antibody response, and the anti-CS GMT of pooled commercial-scale lots was non-inferior to that of a lot formulated from a pilot-scale antigen bulk batch., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The hepatitis E virus (HEV) vaccine, Hecolin(®), was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated HEV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-HEV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin(®)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Schistosoma mansoni tegument is involved in essential functions for parasite survival and represents a target for screening candidates for vaccine and diagnosis. Our group using reverse vaccinology selected six candidates, previously demonstrated by proteomics studies to be expressed in the parasite tegument, among them was Sm200. In this work we have cloned and expressed a recombinant form of Sm200 C-terminal (1069-1520) region. The efficacy of rSm200 (1069-1520) in the diagnosis of schistosomiasis and in the formulation of a vaccine against S. mansoni was assessed respectively in an ELISA based diagnostic assay and immunization protocols in mice. Significant differences between non-infected and acutely infected or chronically infected animals were observed and no cross-recognition was observed with sera from Ascaris suum or Ancylostoma ceylanicum infected mice. rSm200-ELISA test could also discriminate infected individuals from healthy donors not living in endemic area for schistosomiasis but failed to discriminate between individuals from a low endemic area for schistosomiasis known to have positive or negative stools after examination. Recombinant Sm200 also failed to induce protection against schistosomiasis, demonstrating that the C-terminal part of Sm200 is unable to induce protective immune response in mice. Therefore rSm200 (1069-1520)-ELISA represents an important tool to be used in the diagnosis of schistosomiasis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Our work of the last 25 yr was concerned with the development of a vaccine aimed to prevent porcine Taenia solium cysticercosis and was based on cross-reacting Taenia crassiceps antigens that had proved protective against experimental intraperitoneal murine T. crassiceps cysticercosis (EIMTcC). In recent times the efficacy of the vaccine has been considered in need of confirmation, and the use of EIMTcC has been questioned as a valid tool in screening for vaccine candidates among the many antigens possibly involved. A review of our work divided in 2 parts is presented at this point, the first dealing with EIMTcC and the second with porcine T. solium cysticercosis (presented in this issue). Herein, we revise our results using EIMTcC as a measure of the protective capacity of T. crassiceps complex antigen mixtures, of purified native antigens, and of S3Pvac anti-cysticercosis vaccine composed by 3 protective peptides: GK-1, KETc1, and KETc12 either synthetic or recombinantly expressed and collectively or separately, by diverse delivery systems when administered at different doses and by different routes. Statistical analyses of the data lead confidently to the strong inference that S3Pvac is indeed an effective vaccine against EIMTcC via specific and non-specific mechanisms of protection.

Herein we present a review of our research dealing with vaccination against experimental and naturally acquired porcine Taenia solium cysticercosis using Taenia crassiceps-derived antigens. Results strongly support that the different versions of S3Pvac vaccine are indeed effective against porcine T. solium cysticercosis. Immunological results related to vaccination prove that protection is at least partially mediated by specific immunity. The data also support the validity of T. crassiceps murine cysticercosis as an effective tool to identify vaccine candidates against some metacestode infections.

Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

We evaluated the immunogenic and protective potential of a recombinant VapA/CpG oligodeoxynucleotide (ODN) 2395 vaccine in neonatal foals undergoing experimental Rhodococcus equi challenge. Foals (n = 8) were vaccinated by intramuscular injection on days 1 and 15 of the study; control foals (n = 7) received a phosphate-buffered saline (PBS) solution. All foals were challenged by intrabronchial administration of 5 × 10⁶ R. equi 103⁺ on day 29. Bronchoalveolar lavages were done on days 15, 29, and 36 and total cell count, differential cell count, rVapA-stimulated cell proliferation and interferon (IFN)-γ mRNA expression determined. Clinical examination, complete blood (cell) counts, serology for VapA-specific antibodies, and culture of nasal and fecal swabs were done on days 1, 15, 29, 36, 43, and 50. Foals were humanely euthanized on day 50 and severity of pneumonia scored on a 4-point scale. Vaccination resulted in a significant increase in VapA-specific immunoglobulin (Ig) production, with total IgG and IgG(T) being increased by day 15. Expression of VapA-specific IFN-γ mRNA by BAL cells was increased in the vaccinated foals following challenge. Postmortem lung severity scores did not differ between groups. Two foals shed virulent R. equi in feces; however, real-time polymerase chain reaction (PCR) revealed the isolates to be different from the challenge strain.

Background: We have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1-4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP., Methodology/principal Findings: After deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors., Conclusion/significance: All four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia.

Cysteine proteinases 112 (EhCP112) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP112 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 46.29% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP112 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP112 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP112 for human in the future., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Influenza is the most common cause of vaccine-preventable morbidity and mortality despite the availability of the conventional trivalent inactivated vaccine and the live-attenuated influenza vaccine. These vaccines induce an immunity dominated by the response to hemagglutinin (HA) and are most effective when there is sufficient antigenic relatedness between the vaccine strain and the HA of the circulating wild-type virus. Vaccine strategies against influenza may benefit from inclusion of other viral antigens in addition to HA. Epidemiologic evidence and studies in animals and humans indicate that anti-neuraminidase (NA) immunity will provide protection against severe illness or death in the event of a significant antigenic change in the HA component of the vaccine. However, there is little NA immunity induced by trivalent inactivated vaccine and live-attenuated influenza vaccine. The quantity of NA in influenza vaccines is not standardized and varies significantly among manufacturers, production lots and tested strains. The activity and stability of the NA enzyme is influenced by concentration of divalent cations. If immunity against NA is desirable, a better understanding of how the enzymatic properties affect the immunogenicity is needed.

Background: Approximately 35% of the North American population and an estimated 90% of the sub-Saharan African population have antibodies against adenovirus serotype 5 (AdHu5) that are capable of neutralizing AdHu5-based vaccines. In mice, intranasal delivery of AdHu5 expressing the Zaire ebolavirus glycoprotein human adenovirus serotype 5 (Ad) containing the genes for the Zaire ebolavirus glycoprotein (ZGP) under the expressional control of a cytomegalovirus immediate early promoter (CMV)) can bypass systemic preexisting immunity, resulting in protection against mouse-adapted Zaire ebolavirus (Mayinga 1976)., Methods: Guinea pigs administered an adenovirus-based Ebola virus vaccine either intramuscularly or intranasally in the presence of systemically or mucosally induced adenovirus immunity were challenged with a lethal dose of guinea pig-adapted Zaire ebolavirus (Mayinga 1976) (GA-ZEBOV). The humoral immune response was assayed to determine the effect of vaccine delivery route and preexisting immunity., Results: Intramuscular or intranasal vaccination fully protected guinea pigs against a lethal GA-ZEBOV challenge. However, intramuscular vaccination in animals with systemically induced preexisting immunity resulted in low survival following challenge. Interestingly, intranasal vaccination protected guinea pigs with systemic preexisting immunity to AdHu5. Mucosal adenoviral immunity induced by intranasal administration of AdHu5 decreased protection following intranasal vaccination with the first-generation but not with the second-generation vaccine., Conclusions: Intranasal vaccination is an effective vaccine delivery route in the presence of systemic and, to a lower extent, mucosal preexisting immunity to the vaccine vector in guinea pigs.

Background: Outbreaks of filoviral hemorrhagic fever occur sporadically and unpredictably across wide regions in central Africa and overlap with the occurrence of other infectious diseases of public health importance., Methods: As a proof of concept we developed a bivalent recombinant vaccine based on vesicular stomatitis virus (VSV) expressing the Zaire ebolavirus (ZEBOV) and Andes virus (ANDV) glycoproteins (VSVΔG/Dual) and evaluated its protective efficacy in the common lethal Syrian hamster model. Hamsters were vaccinated with VSVΔG/Dual and were lethally challenged with ZEBOV or ANDV. Time to immunity and postexposure treatment were evaluated by immunizing hamsters at different times prior to and post ZEBOV challenge., Results: A single immunization with VSVΔG/Dual conferred complete and sterile protection against lethal ZEBOV and ANDV challenge. Complete protection was achieved with an immunization as close as 3 days prior to ZEBOV challenge, and 40% of the animals were even protected when treated with VSVΔG/Dual one day postchallenge. In comparison to the monovalent VSV vaccine, the bivalent vaccine has slightly reduced postexposure efficacy most likely due to its restricted lymphoid organ replication., Conclusions: Bivalent VSV vectors are a feasible approach to vaccination against multiple pathogens.

Over the next decade, a new generation of vaccines will target the neglected tropical diseases (NTDs). The goal of most NTD vaccines will be to reduce the morbidity and decrease the chronic debilitating nature of these often-forgotten infections &#x2013; outcomes that are hard to measure in the traditional potency testing paradigm. The absence of measurable correlates of protection, a lack of permissive animal models for lethal infection, and a lack of clinical indications that do not include the induction of sterilizing immunity required us to reconsider the traditional bioassay methods for determining vaccine potency. Owing to these limitations, potency assay design for NTD vaccines will increasingly rely on a paradigm where potency testing is one among many tools to ensure that a manufacturing process yields a product of consistent quality. Herein, we discuss the evolution of our thinking regarding the design of a potency assay along these newly defined lines and its application to the release of the experimental Necator americanus-glutathione-S- transferase-1 (Na-GST-1) vaccine to prevent human hookworm infection. We discuss the necessary steps to accomplish the design and implementation of such a new potency assay as a resource for the burgeoning NTD vaccine community. Our experience is that much of the existing information is proprietary and needs to be pulled together in a single source to aid in our overall understanding of potency testing.

Background: Escherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle., Results: Cattle were orally inoculated with either milk (control), milk with live attenuated Salmonella enterica serovar Dublin (vector), or milk with live attenuated recombinant S. Dublin expressing intimin (vaccinated) on days 0, 14 and 28. On day 98, all calves were challenged orally with E. coli O157:H7 to evaluate whether vaccination with the recombinant S. Dublin expressing intimin would reduce the level of E. coli O157:H7 fecal shedding.During the first 28 days, vaccinated calves shed both the vector strain and the intimin-expressing S. Dublin strain at a similar level. The vector strain was shed for a significantly longer period as compared to the level of recombinant vaccine strain. Calves that received the intimin-expressed vaccine ceased shedding S. Dublin from day 28 to day 63. All calves were challenged with E. coli O157:H7 on day 98 to determine the effect on fecal shedding of E. coli O157:H7. The amount of E. coli O157:H7 in feces was measured for 30 days post-challenge. We observed a transient clearance of E. coli O157:H7 from the feces in the vaccinated calves. The magnitude of fecal E. coli O157:H7 shedding did not correlate with the presence of intimin-specific fecal IgA., Conclusion: Oral vaccination with live attenuated recombinant S. Dublin expressing intimin reduced enteric colonization and fecal shedding of E. coli O157:H7. However, the transient clearance of E. coli O157:H7 was not associated with an enhanced IgA-mediated mucosal immune response.

Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity. As a paradigm for peptide vaccine preparation the immunogenic CMV peptide 495-503 NLVPMVATV lyophilisate was resolubilized in dimethyl sulfoxide, phosphate buffered saline and admixed with Montanide. Addition of different amounts of peptide (10-80 microg) to a mixed lymphocyte peptide culture (MLPC) resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8(+) CMV tetramer(+) T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at +/-4 degrees C over 24 h did not result in a significant change of the immunogenicity of the vaccine. In contrast, cryopreservation of the vaccine at -20 degrees C resulted in a loss of immunogenicity. Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as incomplete Freund's adjuvant (IFA), is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.

Background: One strategy for improving anti-tuberculosis (TB) vaccination involves the use of recombinant bacille Calmette-Guérin (rBCG) overexpressing protective TB antigens. rBCG30, which overexpresses the Mycobacterium tuberculosis secreted antigen Ag85b, was the first rBCG shown to induce significantly greater protection against TB in animals than parental BCG., Methods: We report here the first double-blind phase 1 trial of rBCG30 in 35 adults randomized to receive either rBCG30 or parental Tice BCG intradermally. Clinical reactogenicity was assessed, and state-of-the-art immunological assays were used to study Ag85b-specific immune responses induced by both vaccines., Results: Similar clinical reactogenicity occurred with both vaccines. rBCG30 induced significantly increased Ag85b-specific T cell lymphoproliferation, interferon (IFN)-gamma secretion, IFN-gamma enzyme-linked immunospot responses, and direct ex vivo intracellular IFN-gamma responses. Additional flow cytometry studies measuring carboxyfluorescein succinimidyl ester dilution and intracellular cytokine production demonstrated that rBCG30 significantly enhanced the population of Ag85b-specific CD4(+) and CD8(+) T cells capable of concurrent expansion and effector function. More importantly, rBCG30 significantly increased the number of Ag85b-specific T cells capable of inhibiting intracellular mycobacteria., Conclusions: These results provide proof of principal that rBCG can safely enhance human TB immunity and support further development of rBCG overexpressing Ag85b for TB vaccination.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.

A recombinant fowlpox virus (rFPV) expressing the Eimeria tenella rhomboid gene was constructed and its protective efficacy against homologous infection in chickens determined. Three-day-old-specific pathogen free (SPF) chickens were immunized s.c. with 10(2) plaque forming units (PFU), 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, and challenged with 5x10(4) homologous sporulated oocysts 14 days post-immunization (p.i.). The specific antibody response and lymphocyte proliferation were measured 1, 2, 3 and 4 weeks p.i. Oocyst output, body weight gains and lesion scores were measured to evaluate the protective effects of immunization. rFPV-rhomboid elicited a specific humoral immune response and stimulated proliferation of peripheral blood lymphocytes. The lesion scores in groups vaccinated with rFPV-rhomboid were significantly higher than in other groups. At the same time, rFPV-rhomboid improved body weight significantly compared with other groups. Immunization with rFPV-rhomboid reduced oocyst shedding significantly, resulting in a protection rate of 39.6%, 41.1% or 41.7% given a dose of 10(2) PFU, 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, respectively. These results indicated that rFPV can induce immune responses and offer partial protection of chickens against E. tenella challenge.

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n=11) received 12.5 microg of each recombinant protein plus 0.5 microg of cholera toxin, group 2 (G2, n=11) received phosphate buffer saline (PBS) plus 0.5 microg of cholera toxin, and group 3 (G3, n=11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRA7. Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P<0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P>0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T. gondii.

Objective: To evaluate the effectiveness of a recombinant hepatitis B vaccine used in endemic areas of Colombia, as well as risk factors associated with hepatitis B virus (HBV) infection and carriage after vaccine introduction., Methods: A cross-sectional study was carried out in urban and rural areas of the Colombian Amazon, a highly endemic area for hepatitis B infection. Children under 12 years of age and their mothers were selected for the study using one-stage cluster sampling (N=2145) and were examined for HBV serological markers and antibodies against surface antigen (anti-HBs)., Results: There has been a reduction of 60-75% in the prevalence of HBV infection and hepatitis B surface antigen (HBsAg) carriage since HBV vaccination was introduced. Receiving the first dose of HBV vaccine at more than two months after birth was one of the factors associated with HBV carrier status. Maternal HBV infection was also associated with infection in the child., Conclusions: The recombinant Cuban hepatitis B vaccine has contributed to the reduction of the infection in this highly endemic area, though further efforts are required to improve timely vaccination for children at high risk.

After the incursion of bluetongue virus (BTV) into European Mediterranean countries in 1998, vaccination was used in an effort to minimize direct economic losses to animal production, reduce virus circulation and allow safe movements of animals from endemic areas. Vaccination strategies in different countries were developed according to their individual policies, the geographic distribution of the incurring serotypes of BTV and the availability of appropriate vaccines. Four monovalent modified live virus (MLV) vaccines were imported from South Africa and subsequently used extensively in both cattle and sheep. MLVs were found to be immunogenic and capable of generating strong protective immunity in vaccinated ruminants. Adverse side effects were principally evident in sheep. Specifically, some vaccinated sheep developed signs of clinical bluetongue with fever, facial oedema and lameness. Lactating sheep that developed fever also had reduced milk production. More severe clinical signs occurred in large numbers of sheep that were vaccinated with vaccine combinations containing the BTV-16 MLV, and the use of the monovalent BTV-16 MLV was discontinued as a consequence. Abortion occurred in <0.5% of vaccinated animals. The length of viraemia in sheep and cattle that received MLVs did not exceed 35 days, with the single notable exception of a cow vaccinated with a multivalent BTV-2, -4, -9 and -16 vaccine in which viraemia persisted at least 78 days. Viraemia of sufficient titre to infect Culicoides insects was observed transiently in MLV-vaccinated ruminants, and natural transmission of MLV strains has been confirmed. An inactivated vaccine was first developed against BTV-2 and used in the field. An inactivated vaccine against BTV-4 as well as a bivalent vaccine against serotypes 2 and 4 were subsequently developed and used in Corsica, Spain, Portugal and Italy. These inactivated vaccines were generally safe although on few occasions reactions occurred at the site of inoculation. Two doses of these BTV inactivated vaccines provided complete, long-lasting immunity against both clinical signs and viraemia, whereas a single immunization with the BTV-4 inactivated vaccine gave only partial reduction of viraemia in vaccinated cattle when challenged with the homologous BTV serotype. Additional BTV inactivated vaccines are currently under development, as well as new generation vaccines including recombinant vaccines.

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus-Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30-46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development.

The complex process of vaccine product development needs to be tightly controlled and closely monitored to ensure vaccine quality and consistency. Since its inception, PCR has been widely used in all stages of vaccine product development as a tool to assist in the evaluation of vaccine quality, safety and efficacy. In this review, the general principles of conventional and real-time quantitative PCR (Q-PCR) technology and its application in vaccine product development for quantitation of vaccine dose (genome quantitation assay), infectivity (Q-PCR-based potency assay), process residuals, stability, adventitious agents, safety assessment and clinical studies are described. The future outlook and the advantages and disadvantages of this technology are also discussed.

Attenuated live classical swine fever (CSF) viruses are the most efficacious vaccines against the disease. However, little is known about the distribution and detection of CSF vaccine viruses in the host. We therefore compared the new recombinant attenuated marker vaccine virus CP7&#95;E2alf with the conventional C-strain vaccine concerning virus isolation, antigen-, and genome-detection in different samples within the first 42 days post-vaccination (p.v.). Leukocytes and several organs such as tonsils, lymph nodes, spleen, thymus, parotis and kidney were also tested using highly sensitive real-time reverse transcription-polymerase chain reaction (RT-PCR) techniques. It was demonstrated that vaccine virus could be detected by live animal sampling only in a few leukocytes samples at very low titres and genome copy numbers within the first 14 days after immunisation. Vaccine virus could also be isolated from individual tonsil samples within the first 6 days after vaccine application. In contrast, vaccine virus genomes were consistently detected in the tonsils up to day 42 by real-time RT-PCR. Distribution, amount of virus and viral genome levels were similar for both tested vaccines. In conclusion, blood samples could be the sample material of choice for detecting CSF wild type virus infection even in vaccinated animals after more than 14 days p.v., while tonsil sampling provided appropriate material for long-term detection of both tested CSF vaccine viruses using real-time RT-PCR methods.

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.

Sublingual vaccines based on biological extracts from various natural allergen sources are effective in the treatment of respiratory allergies. These vaccines comprise a complex mixture of proteins and glycoproteins that require dedicated standardization procedures to ensure batch-to-batch consistency. Because of the lack of correlation between the potency of an allergen extract and the quantity of major allergen content, standardization is achieved predominantly by determining the global IgE binding capacity of the extract in vitro. New proteomic technologies can be used to further characterize the most abundant proteins present in an extract. Second-generation sublingual vaccines based on recombinant allergens are under development. The aim is to produce molecularly defined vaccines that exhibit superior efficacy, while allowing for simplified immunization schedules. In this approach, recombinant DNA technology is used to express highly purified allergens in their native (i.e. wild-type) conformation. The recombinant allergens are then formulated with ad hoc adjuvants and/or mucoadhesive galenic excipients so that they specifically target oral Langerhans cells and induce allergen-specific regulatory T cells.