WHO Working Group meeting on standardization of acellular pertussis vaccines: Potency assay Beijing, China, 7-9 November 2007.

New acellular pertussis vaccines have recently been developed in China and India. In this context, potency testing and potential improvements of the protective animal models with inclusion of a reference material were recognized as critical issues in the quality assessment of acellular pertussis vaccines. One of these models, namely Modified Intracerebral Challenge Assay (MICA), is currently used as a potency assay in Japan, China and Korea. A collaborative study comparing whole cell references, a candidate acellular pertussis vaccine reference (JNIH-3) and various acellular pertussis products was undertaken in 2006. The results of the collaborative study showed that MICA worked reliably and gave consistent results between laboratories. JNIH-3 was found to give similar dose-response lines to a variety of acellular pertussis vaccines and DTaP formulations, irrespective of the differences in acellular pertussis components. The WHO Working Group agreed that proposal for establishing JNIH-3 as the First International Standard for acellular pertussis vaccine in MICA should be submitted to the Expert Committee on Biological Standardization at its meeting in October 2008.

Intranasal immunization using biphasic lipid vesicles as delivery systems for OmlA bacterial protein antigen and CpG oligonucleotides adjuvant in a mouse model.

The nasal mucosa is an important arm of the mucosal system since it is often the first point of contact for inhaled antigens. The ineffectiveness of the simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies in appropriate delivery systems and adjuvants. We have evaluated biphasic lipid vesicles as a novel intranasal (i.n.) delivery system (designated as vaccine targeting adjuvant, VTA) containing bacterial antigens and CpG oligodeoxynucleotides (ODNs). Results show that administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in greater induction of IgA levels in serum (P< 0.05) and mucosal antibody responses such as IgA in nasal secretions and lung (P< 0.01) after immunization with a combined subcutaneous (s.c.)/i.n. as compared to s.c./s.c. approach. Based on antibody responses, VTA formulations were found to be suitable as delivery systems for antigens and CpG ODNs by the intranasal route, resulting in a Th2-type of immune response, characterized by IgG1 and IL-4 production at the systemic level.

Induction of protective immunity in pigs after immunisation with CpG oligodeoxynucleotides formulated in a lipid-based delivery system (Biphasix).

A large number of studies demonstrated the immunostimulatory effects of CpG oligonucleotides (ODN), particularly in mice. In the present study, we evaluated the ability of lipid-based delivery systems to enhance the adjuvant effect of CpG-ODN and protect against infection in a porcine pleuropneumonia model. Increased levels of OmlA-specific antibody were detected in animals immunised with OmlA and CpG-ODN formulated in the delivery system Biphasix-vaccine targeting adjuvant (VTA), compared to pigs immunised with VTA without CpG-ODN or CpG-ODN alone. In addition, the responses induced by VTA/CpG formulation were similar to those induced by the commercial adjuvant VSA; however, VTA formulations caused significantly less tissue damage than VSA.

Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization.

The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses.

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.

Evaluation of interleukin-4 concentration by ELISA is influenced by the consumption of IL-4 by cultured cells.

The ability to accurately measure cytokine secretion by immune cells is critical to the evaluation of immune mechanisms in the context of Th1 and Th2 responses. In this study, we demonstrated that in vitro consumption of interleukin-4 (IL-4) by stimulated cells influences the concentration of IL-4 in the culture supernatant. In contrast, evaluation of IL-4-secreting cells by ELISPOT is not influenced by the consumption of IL-4 by cultured cells. These discrepancies influence the cytokine profile when responses are evaluated in relation to the secretion of other cytokines, for example, interferon-gamma (IFN-gamma). This information ultimately should enable investigators to evaluate immune responses accurately without concerns of bias resulting from in vitro consumption of IL-4, thus providing much more reliable interpretations of the type of immune response being evaluated.

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle.

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.

Vaccine delivery: lipid-based delivery systems.

Needle-free delivery of vaccines should not only increase compliance, but should also prove to be a safer and less traumatic method of vaccine delivery. One of the potential ways to achieve needle-free delivery is with the use of lipid-based delivery systems. To demonstrate the utility of these systems, we have shown them to be effective with proteins produced by recombinant DNA technology, plasmid-based vaccines, as well as conventional vaccines. Furthermore, these lipid-based delivery systems were shown to be effective in inducing mucosal immunity if delivered to mucosal surfaces or systemic immunity if different transdermally. These approaches have the potential to revolutionize vaccine delivery in humans and animals.

Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery.

Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eradicated. However, despite these successes there are still many microbial diseases that cause tremendous suffering because there is no vaccine or the vaccines available are inadequate. In addition, even if vaccines were available for all infectious diseases there is no guarantee that people would use them routinely. One of the major impediments to ensuring vaccine efficacy and compliance is that of delivery. Presently most vaccines are given by intramuscular administration. Unfortunately this is often traumatic, especially in infants. Thus, if it was possible to replace intramuscular immunization by mucosal (oral/intranasal) or transdermal delivery it may be possible to both enhance mucosal immunity as well as improve overall compliance rates. The transdermal route has been used by the pharmaceutical industry for the delivery of various low molecular weight drugs. Some of the approaches used for smaller compounds may also have potential for delivery of either protein or polynucleotide vaccines. However, there is a greater challenge to delivering large molecular weight molecules through the skin due to size, charge and other physicochemical properties. This review will describe the recent advances that have been made in dermal and topical delivery as related to vaccines.

Effects of IL-12 on immune responses induced by transcutaneous immunization with antigens formulated in a novel lipid-based biphasic delivery system.

The development of non-invasive methods for the delivery of proteins through the permeability barriers, such as the intact skin, will greatly facilitate the administration of human and veterinary vaccines. In the present study we used recombinant Pasteurella haemolytica leukotoxin (Lkt) and hen egg lysozyme (HEL) as model antigens to investigate the ability of transdermal administration of vaccine antigens to induce humoral and cellular responses in mice and to assess the immunomodulatory effects of IL-12 on these antigen-specific immune responses. Mice were immunized by the transdermal route with Lkt or HEL formulated in a novel lipid-based biphasic delivery system (BPDS). Transdermal delivery of Lkt or HEL induced strong polarized Th2 responses characterized by enhancement of antigen-specific IgG1 antibody subclass and predominant induction of antigen specific IL-4 over IFN-gamma in spleen and draining lymph nodes cells. Animals immunized by topical application of formulations containing antigen and IL-12 developed significantly lower antibody titres without significant changes in IL-4 or IFN-gamma secreting cells (SC) in the draining lymph nodes or spleen cells. Our results indicated that application of antigens formulated in BPDS induced antigen-specific immune responses. Furthermore, incorporation of IL-12 to the vaccine formulation influences the induction of antibody responses induced by transdermal immunization. We demonstrated the feasibility of using this technology for the development of non-invasive methods of vaccine administration.

Transtracheal administration of interleukin-12 induces neutrophil responses in the murine lung.

Although the roles of interleukin-12 (IL-12) in the immunomodulation of antigen-specific responses are well characterized, the effects of IL-12 on the respiratory tract following mucosal administration are not well defined. Therefore, we investigated changes in the murine lung shortly after intranasal (i.n.) administration of murine IL-12. We showed that IL-12 induced neutrophil influx to the murine lung in both C57BL/6 and BALB/c mice. Histologic examination revealed that intranasal administration of IL-12 with liposomes induced focal neutrophil infiltration into the alveoli and a significant increase in neutrophils in bronchoalveolar lavage fluids when compared with administration of liposomes alone. In vitro chemotaxis assays indicated that the observed pulmonary neutrophil response induced by IL-12 could have been due in part to the direct chemotactic activity of IL-12 for murine neutrophils.

Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses.

The induction of mucosal immune responses by a liposome-formulated Y. pestis vaccine (formaldehyde-killed whole cell vaccine; KWC) was evaluated. We demonstrated that intranasal immunization of mice with Y. pestis KWC vaccine, formulated with liposomes, significantly enhanced mucosal immune responses in the lung when compared to the responses induced with KWC vaccine alone. These immune responses were characterized by increased titres of specific IgA and IgG in mucosal secretions (lung and nasal washes), and an increased frequency of specific antibody-secreting cells in the lungs. In addition, antigen-specific proliferative responses and IFN-gamma-secreting cells were also significantly enhanced in the spleens of mice immunized with the KWC vaccine formulated in liposomes. Animals that were immunized intranasally with the KWC vaccine showed significant protection against an intranasal challenge with Y. pestis. These results highlight the importance of mucosal administration of vaccine antigens to stimulate immunity in the respiratory tract and demonstrate that liposome formulations can improve the effectiveness of conventional vaccines.

Induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector.

We investigated the antigen-specific mucosal and systemic immune responses of newborn lambs following enteric immunization, targeting jejunal Peyer's patches with a human adenovirus vector that expressed the glycoprotein D (gD) of bovine herpesvirus-1. Both humoral and cell-mediated gD-specific mucosal immune responses were detected in newborn lambs (1-4 days old) after a single immunization and these responses were qualitatively and quantitatively similar to those detected in 5-6-week-old lambs. Passively transferred gD-specific maternal antibody did not significantly alter either mucosal or systemic gD-specific immune responses. Furthermore, enteric immunization of newborn lambs primed mucosal immune responses in the lungs. These observations confirmed that gut-associated lymphoid tissue of a newborn ruminant is immune competent and that enteric immunization may be an effective approach for the induction of both mucosal and systemic immune responses in the neonate.

Dermal and transdermal delivery of protein pharmaceuticals: lipid-based delivery systems for interferon alpha.

The dermal and transdermal delivery of protein pharmaceuticals faces enormous challenges, and at the same time has very significant potential for the non-invasive treatment of both localized and systemic diseases. In this article we review the various approaches used to enhance and control the delivery of protein therapeutic agents through the dermal barrier. We show results of the delivery of interferon (IFN) alpha, an antiviral agent used in the treatment of condylomata acuminata (genital warts), using lipid-based delivery systems (LBDS). In the general category of LBDS, we investigated the use of liposomes and fatty acylation as ways to increase IFNalpha delivery into human skin.

Polynucleotide vaccines: potential for inducing immunity in animals.

Polynucleotide immunization has been described as the Third Revolution in Vaccinology. Early studies suggest the potential benefits of this form of immunization including: long-lived immunity, a broad-spectrum of immune responses (both cell mediated immunity, and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using a murine model, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to mucosal surfaces have been attempted and are described. Expansion of studies in various species, using natural models, should be extremely helpful in demonstrating the universality of this approach to immunization and more importantly, accurately identify parameters that are critical for the development of protective immunity.

Ileal and jejunal Peyer's patches play distinct roles in mucosal immunity of sheep.

The majority of pathogens enter the body through mucosal surfaces and it is now evident that mucosal immunity can provide effective disease protection. However, the induction of mucosal immunity will require efficient targeting of mucosal vaccines to appropriate mucosa-associated lymphoid tissue. An animal model, based upon the surgical preparation of sterile intestinal 'loops' (blind-ended segments of intestine), was developed to evaluate mucosal and systemic immune responses to enteric vaccines in ruminants. The effectiveness of end-to-end intestinal anastomoses was evaluated and fetal surgery did not disrupt normal intestinal function in lambs up to 6-7 months after birth. The immunological competence of Peyer's patches (PP) within the intestinal 'loops' was evaluated with a human adenovirus 5 vector expressing the gD gene of bovine herpesvirus-1. This vaccine vector induced both mucosal and systemic immune responses when injected into intestinal 'loops' of 5-6-week-old lambs. Antibodies to the gD protein were detected in the lumen of intestinal 'loops' and serum and PP lymphocytes proliferated in response to gD protein. The immune competence of ileal and jejunal PP was compared and these analyses confirmed that jejunal PP are an efficient site for the induction of mucosal immune responses. This was confirmed by the presence of gD-specific antibody-secreting cells in jejunal but not ileal PP. Systemic but not mucosal immune responses were detected when the vaccine vector was delivered to the ileal PP. In conclusion, this model provided an effective means to evaluate the immunogenicity of potential oral vaccines and to assess the immunological competence of ileal and jejunal Peyer's patches.

Antigen-specific cytokine and antibody isotype profiles induced by mucosal and systemic immunization with recombinant adenoviruses.

We investigated antigen-specific antibody and T-cell responses in mice immunized with human adenovirus type 5 (HAd5) vectors expressing either the authentic or truncated form of glycoprotein D (gD and tgD, respectively) of bovine herpesvirus type 1 (BHV-1). We also tested whether different routes of immunization influenced the level and type of immunity. Immunization intranasally (i.n.) stimulated higher levels of gD-specific IgA in the lung and nasal washes and induced a higher frequency of gD-specific antibody secreting cells (CSs) in the lung than did immunization subcutaneously (s.c.). In addition, immunization i.n. stimulated gD-specific systemic antibody responses of a higher IgG1/IgG2a ratio and lower numbers of gD-specific interferon (IFN)-gamma SCs in the spleen than did immunization s.c. HAd5-specific responses also depended on the route of immunization and were characterized by lower IFN-gamma interleukin (IL)-4 ratios than gD-specific responses. Immunization with the tgD-expressing vector induced generally lower antibody and cytokine responses than the gD-expressing vector. Higher numbers of antigen-specific IgA SCs in the lung as measured by enzyme-linked immunospot (ELISPOT) assay correlated with higher levels of IgA in the respiratory tract as measured by enzyme-linked immunosorbent (ELISA) assay, although there was no such correlation for IgG responses of any isotype. In conclusion, the route of immunization and form of antigen had an impact on the level and type of immune responses induced by adenovirus vectors.

Induction of mucosal immune responses by administration of liposome-antigen formulations and interleukin-12.

We examined the effect of interleukin-12 (IL-12) on the induction of mucosal immune responses following intranasal immunization with liposome-antigen formulations. We assessed the immune response to two recombinant glycoproteins (gD and gB) from bovine herpesvirus type 1 (BHV-1). Positively charged liposomes induced significantly higher gD-specific IgA titers than did immunization with antigen alone. This liposome formulation was selected to further assess the ability of IL-12 to influence mucosal immune responses. Intranasal immunization with IL-12 gD-liposome formulations did not alter the induction of mucosal immune responses. However, a significant increase in anti-gD antibody responses was induced in serum after intranasal immunization with IL-12 gD-liposome when compared with animals immunized with gD-liposomes. Mucosal antibody responses induced by a subcutaneous priming followed by an intranasal boost were significantly higher than those induced by two intranasal immunizations with the same IL-12 liposome-gD formulations. Furthermore, this immunization protocol resulted in the induction of high levels of interferon-gamma (IFN-gamma) in the lungs of subcutaneously primed mice. These findings indicate that the immunomodulatory effects of IL-12 influenced immune responses to a vaccine antigen when delivered intranasally and that these responses can be further enhanced by subcutaneous priming.

Mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1.

To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.

The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.

We investigated whether pre-existing adenovirus-specific immunity influenced the development of immunity to a foreign antigen expressed by recombinant adenovirus. Active adenovirus-specific immunity was induced in cotton rats by i.n. administration of wild type human adenovirus type 5 (HAd5) two weeks before immunisation with a HAd5 vector expressing the glycoprotein D (gD) of bovine herpesvirus type 1 (gD-dE3 recombinant adenovirus). Active adenovirus-specific immunity inhibited gD-specific immune responses, following either i.n. or gastrointestinal immunisation with gD-dE3. An inhibitory effect was present even if infection with HAd5 and immunisation with gD-dE3 were 13 weeks apart. Passive transfer of adenovirus specific antibodies to cotton rats one day before immunisation, however, did not significantly inhibit gD-specific immune responses induced by i.n. immunisation with gD-dE3. Repeated administration of an adenovirus vector, therefore, may have a limited ability to deliver antigen, while passive immunity to adenovirus may not interfere with the success of immunisation.