Mutant Escherichia coli heat-labile enterotoxin [LT(R192G)] enhances protective humoral and cellular immune responses to orally administered inactivated influenza vaccine

Lu, X., J. D. Clements, and J. M. Katz
Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA

Influenza vaccines capable of inducing both systemic and mucosal antibody responses are highly desirable. Optimal induction of mucosal IgA is accomplished by mucosal delivery of vaccine. Mucosal adjuvants may improve the immunogenicity and efficacy of vaccines delivered by this route. Here, we compare the adjuvant activities of a mutant of heat-labile enterotoxin from Escherichia coli [LT(R192G)] with those of the wildtype LT (wtLT) for oral vaccination with inactivated influenza vaccine in BALB/c mice. Compared with administration of oral influenza vaccine alone, co-administration of vaccine with LT(R192G) provided enhanced protection from infection in the upper and lower respiratory tract equivalent to and at similar doses as that obtained with wtLT. Likewise, LT(R192G) augmented virus-specific IgG and IgA responses in serum, lung and nasal washes and the numbers of virus-specific antibody-forming cells in spleen, lung and Peyer’s patches in a manner comparable to wtLT. Virus-specific splenic CD4+ cells from mice administered oral vaccine with either adjuvant produced a mixed Th1- and Th2-type cytokine response pattern. Taken together, these results indicate that LT(R192G), like wtLT, is a potent adjuvant for oral vaccination of mice with influenza vaccine.

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Intranasal immunotherapy for the treatment of Alzheimer's disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants.

Lemere, C. A., E. T. Spooner, J. F. Leverone, C. Mori, and J. D. Clements
Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.

Alzheimer’s disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure. Extracellular deposition of amyloid-β protein (A β) in brain is a key neuropathological characteristic of AD. In 1999, Schenk et al. first reported that an injected A β vaccine given to PDAPP mice, an AD mouse model displaying A β deposition in brain, led to the lowering of A β levels in brain. In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic A β 1–40 peptide for 7 months led to a 50–60% reduction in cerebral A β burden in PDAPP mice; serum A β antibody titers were low (~26µg/ml). More recently, we have optimized our i.n. A β immunization protocol in wild-type (WT) mice. When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with A β i.n., there was a dramatic 12-fold increase in A β antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n. A β without adjuvant. A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-A β antibody titers were 16-fold higher than those seen in mice given i.n. A β without adjuvant. In both cases, the serum A β antibodies recognized epitopes within A β 1–15 and were of the immunoglobulin (Ig) isotypes IgG2b, IgG1, IgG2a and low levels of IgA. This new and improved A β vaccine protocol is now being tested in AD mouse models with the expectation that higher A β antibody titers may be more effective in reducing cerebral A β levels.

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Induction of serum and mucosal FIV-specific immune responses by intranasal immunization with p24Gag

Leavell, S., B. Wright, L. Scappino, J. Sirriyah, C. Chen, J. D. Clements, and M. J. Burkhard
Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA

We examined the ability of FIV p24Gag to induce systemic and mucosal FIV-specific immune responses when delivered as a nasal immunogen alone, or with a mucosal adjuvant, Escherichia coli heat labile toxin LT(R192G). Nasal immunization with p24Gag alone induced FIV-specific immune responses but overall responses were weak, transient, and/or present only in a few animals. Co-administration of LT(R192G) resulted in strong FIV-specific serum IgG and enhanced salivary IgA responses. Moreover, FIV-specific IgA was detected in vaginal wash fluid from 6/6 cats co-immunized with LT(R192G) and p24Gag versus 1/6 immunized with p24Gag alone. This is the first report detailing induction of systemic or mucosal FIV-specific immune responses by nasal immunization alone. As such, this study demonstrates that nasal immunization of cats can be a relevant and effective route for the delivery of candidate vaccines. However, while nasal immunization of cats with p24Gag induces antigen-specific systemic immune responses, development of strong systemic and mucosal immune responses requires co-administration of a mucosal adjuvant, such as LT(R192G).

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Effect of homologous and heterologous prime-boost on the immune response to recombinant plague antigens

Glynn A., L. C. Freytag, J. D. Clements
Department of Microbiology and Immunology, Tulane University, School of Medicine, New Orleans, Louisiana 70112, USA

Among the pathogens that have been identified as potential agents of biological warfare or bioterrorism, Yersinia pestis is one of the main concerns due to the severity and potential transmissibility of the pneumonic form of the disease in humans. There are no approved vaccines for protection against pneumonic plague, but a Y. pestis-derived fusion protein (F1–V) has shown great promise as a protective antigen in murine studies. In the current study, we examine different prime–boost regimens, including parenteral, mucosal, and transcutaneous delivery, in order to explore the effect of changing the route of prime and boost on the ability of recombinant F1–V to promote the development of long-lasting, high-titer antibodies. The most significant findings of the study reported here are that (1) intranasal and subcutaneous immunizations are both effective and essentially equivalent for induction of serum and bronchioalveolar anti-F1–V IgG1 responses when a single booster dose is administered by the same (homologous) route, (2) heterologous boosting can be as or more effective than homologous boosting for induction of either serum or bronchioalveolar anti-F1–V IgG1 responses, and (3) anti-F1 and anti-V total IgG responses were highest in animals primed intranasally and boosted by any route when compared to animals primed transcutaneously or subcutaneously. As with previously published studies, there were still significant levels of circulating anti-F1–V antibodies 1 year post-primary immunization. These studies provide important insights into the development of new-generation biodefense vaccines.

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Protection against aerosolized Yersinia pestis challenge following homologous and heterologous prime-boost with recombinant plague antigens

Glynn, A., C. J. Roy, B. S. Powell, J. J. Adamovicz, L. C. Freytag, and J. D. Clements
Department of Microbiology and Immunology, Tulane University, School of Medicine, New Orleans, Louisiana 70112, USA

A Yersinia pestis-derived fusion protein (F1–V) has shown great promise as a protective antigen against aerosol challenge with Y. pestis in murine studies. In the current study, we examine different prime–boost regimens with F1-V and demonstrate that (1) boosting by a route other than the route used for the priming dose (heterologous boosting) protects mice as well as homologous boosting against aerosol challenge with Y. pestis, (2) parenteral immunization is not required to protect mice against aerosolized plague challenge, (3) the route of immunization and choice of adjuvant influence the magnitude of the antibody response as well as the IgG1/IgG2a ratio, and (4) inclusion of an appropriate adjuvant is critical for non-parenteral immunization.

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