Lessons along the Critical Path: Developing Vaccines against Human Helminths.

Helminthic parasites are important targets for vaccine research as they infect an estimated 1 billion people worldwide. Despite significant progress in the discovery of defined antigens as candidates for vaccines, the potential of a helminth vaccine advancing to an investigational product to be tested in humans remains as challenging as it did 50 years ago. Candidate helminth vaccines must still advance along a 'critical path' of preclinical research, vaccine process development (which includes 'chemistry, manufacturing, and controls' or CMC), current good manufacturing practice (cGMP) production of the vaccine, and clinical trials. This path is highly targeted towards meeting the safety, immunogenicity, and efficacy criteria of regulatory bodies such as the US Food and Drug Administration (FDA). For nearly 20 years our product development partnership (PDP), the Texas Children's Hospital Center for Vaccine Development (TCH-CVD), has followed the critical paths of several novel subunit vaccines for the human hookworm Necator americanus and the intestinal trematode Schistosoma mansoni. Herein, we describe the critical lessons learned along this critical path.

A randomized lot-to-lot immunogenicity consistency study of the candidate zoster vaccine HZ/su.

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).

Particle and subunit-based hemagglutinin vaccines provide protective efficacy against H1N1 influenza in pigs.

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.

Development and validation of glycoprotein-based native-subunit vaccine for fish against Aeromonas hydrophila.

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein-based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA-specific lectin and size-exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3 ] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.

Multiantigenic subunitary vaccines against tuberculosis in clinical trials: Where do we stand and where do we need to go?

The idea of presenting this commentary is to bring attention to the current status of clinical tests from several multiantigen vaccine candidates based on proteins produced by means of genetic engineering and molecular biology approaches and to suggest how new emerging technologies (OMICs) and bioinformatics might benefit vaccine development for better control of tuberculosis.

Current Progress in Developing Subunit Vaccines against Enterotoxigenic Escherichia coli-Associated Diarrhea.

Diarrhea continues to be a leading cause of death in children <5 years of age, and enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of children's diarrhea. Currently, there are no available vaccines against ETEC-associated diarrhea. Whole-cell vaccine candidates have been under development but require further improvements because they provide inadequate protection and produce unwanted adverse effects. Meanwhile, a newer approach using polypeptide or subunit vaccine candidates focusing on ETEC colonization factor antigens (CFAs) and enterotoxins, the major virulence determinants of ETEC diarrhea, shows substantial promise. A conservative CFA/I adhesin tip antigen and a CFA MEFA (multiepitope fusion antigen) were shown to induce cross-reactive antiadhesin antibodies that protected against adherence by multiple important CFAs. Genetic fusion of toxoids derived from ETEC heat-labile toxin (LT) and heat-stable toxin (STa) induced antibodies neutralizing both enterotoxins. Moreover, CFA-toxoid MEFA polypeptides, generated by fusing CFA MEFA to an STa-LT toxoid fusion, induced antiadhesin antibodies that broadly inhibited adherence of the seven most important ETEC CFAs associated with about 80% of the diarrhea cases caused by ETEC strains with known CFAs. This same antigen preparation also induced antitoxin antibodies that neutralized both toxins that are associated with all cases of ETEC diarrhea. Results from these studies suggest that polypeptide or subunit vaccines have the potential to effectively protect against ETEC diarrhea. In addition, novel adhesins and mucin proteases have been investigated as potential alternatives or, more likely, additional antigens for ETEC subunit vaccine development.

Protective immunity against Eimeria tenella infection in chickens induced by immunization with a recombinant C-terminal derivative of EtIMP1.

Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. Cloning and sequence analysis has predicted the antigen to be a novel membrane protein of apicomplexan parasites. In order to assess the immunogenicity of EtIMP1, a C-terminal derivative of EtIMP1 was expressed in a bacterial host system and was used to immunize chickens. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the subunit vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. The results suggested that the C-terminal derivative of EtIMP1 might be considered as a candidate in the development of subunit vaccines against Eimeria infection.

Safety and immunogenicity of influenza A H5 subunit vaccines: effect of vaccine schedule and antigenic variant.

BACKGROUND: The current US national stockpile of influenza H5 vaccine was produced using the antigen from the strain A/Vietnam/1203/2004 (a clade 1 H5 virus). Recent H5 disease has been caused by antigenically divergent H5 viruses, including A/Indonesia/05/2005 (a clade 2 H5 virus). METHODS: The influence of schedule on the antibody response to 2 doses of H5 vaccines (one a clade 1 hemagglutinin protein [HA] vaccine and one a clade 2 HA vaccine) containing 90 µg of antigen was evaluated in healthy adults 18-49 years of age. RESULTS: Two doses of vaccine were required to induce antibody titers ≥ 1:10 in most subjects. Accelerated schedules were immunogenic, and antibody developed after vaccinations on days 0 and 7, 0 and 14, and 0 and 28, with the day 0 and 7 schedule inducing lower titers than those induced with the other schedules. With mixed vaccine schedules of clade 1 followed by clade 2 vaccine administration, the first vaccination primed for a heterologous boost. The heterologous response was improved when the second vaccination was given 6 months after the first, compared with the response when the second vaccination was given after an interval of 1 month. CONCLUSIONS: An accelerated vaccine schedule of injections administered at days 0 and 14 was as immunogenic as a vaccine schedule of injections at days 0 and 28, but both schedules were inferior to a vaccine schedule of injections administered at 0 and 6 months for priming for heterologous vaccine boosting. Clinical Trial Registry Number: NCT00703053.

Cytomegalovirus vaccination of leukemia and lymphoma patients after allogeneic stem cell transplantation--validation of a peptide vaccine.

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.

A peptide vaccine administered transcutaneously together with cholera toxin elicits potent neutralising anti-FMDV antibody responses.

In this study a synthetic peptide representing residues 141-159 from the GH loop of VP1 protein of foot-and-mouth disease virus was tested for its capacity to elicit virus neutralising antibodies in mice after transcutaneous immunisation. Topical application of the peptide conjugated to bovine serum albumin together with cholera toxin as an adjuvant elicited anti-peptide antibody responses with strong virus neutralising activity. The combination of cholera toxin with an immunostimulatory CpG motif resulted in the induction of IgG1 and IgG2a anti-peptide antibodies with significantly enhanced virus neutralising activity. To shed more light on the mechanisms of cholera toxin adjuvanticity we demonstrated its binding to keratinocytes via GM(1)-gangliosides. This was followed by an increase of the intracellular cAMP and the rapid diffusion of cholera toxin throughout the epidermis. These findings demonstrate that peptide-based vaccines when combined with the appropriate adjuvant(s) can elicit potent virus neutralising antibody responses after transcutaneous immunisation. However, experiments in target species will be required to confirm the potential of this simple vaccination procedure in livestock.

Development of a hepatitis C virus vaccine.

Any program aimed at the development of a vaccine should consider several important issues because they may greatly influence the choice of immunogen used in the vaccine, the delivery system selected for its application, the population to be vaccinated, and the type of vaccine to be developed (ie, preventive or therapeutic). These issues concern the epidemiology of the infectious disease targeted, the actual routes of transmission, the antigenic diversity of the infectious agent, the existing therapies, and their rate of success. In the case of hepatitis C virus, a viral agent whose clinical existence was recognized in the 1970s but which was only identified by the use of molecular cloning technology in the late 1980s, some of these issues are particularly relevant.

Preliminary approach to find synthetic peptides from nodavirus capsid potentially protective against sea bass viral encephalopathy and retinopathy.

Four synthetic peptides of 15 amino acids (aa), corresponding to sequences of the nodavirus DIEV RNA(2) protein, were chosen to test their potential immunogenicity in sea bass. Two of these included the N or C terminal regions (N-ter or C-ter) and the sequences of the others contained a potential external site (aa 127-140: Lp1 and as 266-279: Lp2). Two heat inactivated strains of nodavirus (HI Sb1 and HI Sb2), were used as positive controls and the carrier (KLH) as a negative control. ELISAs were performed to quantify serum antibodies specific to nodavirus, to peptides, and to the carrier in order to monitor their immunogenicity. All the fish reacted to the peptides C-Ter, Lp1 and Lp2 but only 55% of animals injected with N-ter produced specific antibodies. The proportion of fish that produced antibodies that cross reacted with nodavirus was very different with regard to the antigen injected: HI Sb1=88%; HI Sb2=85%; N-ter=38%; C-ter=27%. Protection against nodavirus was investigated by challenging the fish with a virulent viral suspension. The results showed that heat-inactivated Nodavirus protect fish and the N-ter peptide is a potential protective peptide. This initial approach showed that although vaccinating fish with peptides is possible, the tools and strategies of the research used in this field still need to be adapted to fish.

Improved immunogenicity of DNA vaccination with mycobacterial HSP65 against bovine tuberculosis by protein boosting.

A scientific review for the government of the United Kingdom has recommended that the development of a cattle vaccine against bovine tuberculosis holds the best prospects to control this disease in the national herd. As BCG vaccination of cattle results in variable degrees of protection, novel vaccine strategies that could replace or supplement BCG are required. In this study, the mycobacterial antigen HSP65 was used to determine whether priming cattle with a plasmid DNA vaccine and subsequently boosting with the recombinant protein in adjuvant (heterologous prime-boost approach) would result in improved and more homogenous immune responses over immunising with plasmid DNA or protein in adjuvant alone. The results demonstrated that strong, and compared to protein or DNA vaccination protocols alone, more homogenous, cellular immune responses were induced in cattle vaccinated with the prime-boost regimen. In addition, DNA prime-protein boost vaccination as well as protein vaccination resulted in stronger humoral immune responses with a balanced IgG profile compared to DNA vaccination alone. Importantly, none of the vaccination protocols sensitised cattle to the intradermal tuberculin test suggesting that TB subunit vaccines can be designed to allow the continued use of the tuberculin test to discriminate between vaccinated cattle and those infected with Mycobacterium bovis.

Effects of active immunization against GnRH on serum LH, inhibin A, sexual development and growth rate in Chinese female pigs.

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan x Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10-13 weeks. One pig from each litter was immunized with 62.5 microg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P < 0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3 +/- 0.2 and 43.9 +/- 11.4 g, respectively; mean +/- S.E.M.) were significantly lower (P < 0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P < 0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74 +/- 0.04 versus 0.66 +/- 0.04 versus 0.66 +/- 0.03 kg per day, respectively; mean +/- S.E.M., P = 0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.

Development of cytomegalovirus vaccines: prospects for prevention of congenital CMV infection.

Congenital cytomegalovirus (CMV) infection is an important cause of hearing, cognitive, and motor impairments that cannot be effectively prevented or treated by any current medical or public health interventions. A review of priorities for vaccine development by The Institute of Medicine of the National Academy of Sciences concluded that a vaccine to prevent congenital CMV infection should be a top priority for the United States. Evidence from clinical studies indicates that immunity to CMV can reduce the frequency and severity of disease. Laboratory investigations have identified structural and nonstructural CMV proteins that play a key role in eliciting protective immunity. The rationale for development of a CMV vaccine has been strengthened further by studies in experimental animals demonstrating the ability of immunization with subunit vaccines to prevent disease and transplacental transmission of virus. At least 4 CMV vaccines are in clinical trials, and advances in biotechnology are paving the way for additional novel vaccines.

Lipopeptide vaccines--yesterday, today, and tomorrow.

Peptide-based vaccines offer several potential advantages over the conventional whole proteins (or whole gene, in the case of genetic immunisation) in terms of purity and a high specificity in eliciting immune responses. However, concerns about toxic adjuvants, which are critical for immunogenicity of synthetic peptides, still remain. Lipopeptides, a form of peptide vaccine, discovered more then a decade ago, are currently under intensive investigation because they can generate comprehensive immune responses, without the use of adjuvants. In this review, we address the past of lipopeptide vaccines, highlight the progress made toward their optimisation, and stress future challenges and issues related to their synthesis, formulation, and delivery. In particular, the recent development of mucosal application of lipopeptide vaccines may present an ideal strategy against many pathogens that infect mucosal surfaces.

Vaccination against the HER-2/neu oncogenic protein.

The HER-2/neu oncogenic protein is a well-defined tumor antigen. HER-2/neu is a shared antigen among multiple tumor types. Patients with HER-2/neu protein-overexpressing breast, ovarian, non-small cell lung, colon, and prostate cancers have been shown to have a pre-existent immune response to HER-2/neu. No matter what the tumor type, endogenous immunity to HER-2/neu detected in cancer patients demonstrates two predominant characteristics. First, HER-2/neu-specific immune responses are found in only a minority of patients whose tumors overexpress HER-2/neu. Secondly, immunity, if detectable, is of low magnitude. These observations have led to the development of vaccine strategies designed to boost HER-2/neu immunity in a majority of patients. HER-2/neu is a non-mutated self-protein, therefore vaccines must be developed based on immunologic principles focused on circumventing tolerance, a primary mechanism of tumor immune escape. HER-2/neu-specific vaccines have been tested in human clinical trials. Early results demonstrate that significant levels of HER-2/neu immunity can be generated with active immunization. The T-cell immunity elicited is durable after vaccinations have ended. Furthermore, despite the generation of CD8(+) and CD4(+) T-cells responsive to HER-2/neu in a majority of patients, there is no evidence of autoimmunity directed against tissues that express basal levels of the protein. Cancer vaccines targeting the HER-2/neu oncogenic protein may be useful adjuvants to standard therapy and aid in the prevention of relapse in patients whose tumors overexpress the protein. Furthermore, boosting HER-2/neu-specific T-cell frequencies via active immunization may allow the ex vivo expansion of HER-2/neu-specific T-cells for use in adoptive immunotherapy, a therapeutic strategy directed against the treatment of established disease.

Vaccination of calves against bovine herpesvirus-1: assessment of the protective value of eight vaccines.

Eight separate, but related experiments, were carried out in which groups of six calves were vaccinated with one of eight commercial vaccines. In each experiment the vaccinated calves were subsequently exposed to three calves infected with virulent bovine herpesvirus-1 (BHV-1). In each experiment, all infected donor calves developed a typical severe infectious bovine rhinotracheitis (IBR) infection and excreted virus in their nasal secretions of up to 10(8.00) TCID50/0.1 ml. One live BHV-1 gE-negative vaccine (A) and three modified live vaccines (B, C, D), administered intranasally, all protected against clinical disease. The calves vaccinated with one vaccine (C) also did not excrete virus in the nasal secretions, whereas the calves protected by vaccines A, B and D excreted virus in their nasal secretions but at low titres (10(0.66)-10(1.24) TCID50/0.1 ml). A fourth modified live vaccine (E), given intramuscularly, failed to prevent mild clinical disease in the calves which also excreted virus in the nasal secretions at titre of 10(1.00) TCID50/0.1 ml. An analogous result was given by the calves vaccinated with either of the two inactivated vaccines (F and G) or with a BHV-1 subunit vaccine (H). All calves developed mild clinical signs and excreted virus at titres of 10(2.20)-10(3.12) TCID50/0.1 ml. Calves vaccinated with C vaccine were subsequently given dexamethasone, following which virus was recovered from their nasal secretions. The virus isolates did not cause disease when calves were infected and appeared to be closely related to the vaccine strain.