Our study is the first conducted in humans of an intranasal vaccine with LTK63 as an adjuvant. We showed that intranasal influenza vaccines with LTK63 as an adjuvant and an intramuscular vaccine with MF59 as an adjuvant stimulate different antibody responses, which is consistent with comparisons of live ca
influenza vaccine and plain, inactivated vaccine (5
). The strongest virus-specific mucosal IgA responses in our study occurred with the intranasal vaccine containing 30 μg LTK63 and biovector, which significantly increased IgA levels to all three virus strains. In contrast, two doses of intramuscular vaccine with MF59 as an adjuvant failed to elicit significant rises in mucosal IgA to any virus strain. Virus-specific IgA mucoconversion rates (i.e., ≥2.5-fold increases in IgA antibody concentration) for A/Panama/2007/99 (H3N2), A/Duck/Singapore/97 (H5N3), and B/Guandong/2000 ranged from 47% to 93% in those given the vaccine containing 30 μg LTK63 and biovector, and the mean virus-specific IgA levels increased 2.8- to 6.3-fold. The addition of the biovector enhanced the mucosal IgA responses to two of the three vaccine strains. The IgA response to the intranasal vaccine containing 30 μg LTK63 and biovector is comparable with that of an inactivated intranasal virosomal vaccine, which was licensed in Switzerland for the 2000-2001 influenza season and contained wild-type E. coli
heat-labile holotoxin as a mucosal adjuvant—the corresponding IgA mucoconversion rates for the A-H1N1, A-H3N2, and influenza B virus antigens in the Swiss vaccine were 50 to 57%, and geometric mean titers increased 2.5- to 2.8-fold compared to the baseline (13
). Similarly, the IgA responses in our study elicited to the vaccine formulation containing 30 μg LTK63 and biovector are comparable with those elicited by the live ca
influenza vaccine that is licensed in the United States, as roughly one-half of recipients given live vaccine respond with local IgA production to influenza A H3N2 and A H1N1 viruses, while the response to influenza B virus is evidently lower (5
). For reasons that are unclear, we found that the serum hemagglutination inhibition responses and mucosal IgA responses to currently circulating virus strains in intranasally delivered vaccine formulations differed by the strain of influenza virus.
The percentages of subjects with a fourfold rise in HAI titer (seroconversion) after two doses of vaccine with 30 μg LTK63 and biovector were 27% for A/Panama (H3N2) and 67% for B/Guandong virus. As expected, the HAI results showed that participants were more likely to seroconvert if they received the parenteral vaccine than if they received the intranasal vaccine, which is consistent with such comparisons with live intranasal vaccine or inactivated intranasal vaccine containing E. coli
heat-labile holotoxin (5
). This effect is more pronounced in populations with low or absent prevaccination antibody than in those with various degrees of seropositivity prior to vaccination (5
) and was evident in our study, where the neutralizing antibody response to A/Duck/Singapore/97 (H5N3) by recipients of the intranasal vaccine was no better than that to the placebo. Nonetheless, although the number of participants was small, we showed that the intranasal formulation containing 30 μg LTK63 and biovector fulfilled all three CHMP criteria for the assessment of interpandemic vaccines, when assessed at six weeks, for the B/Guandong component. The measurement of serum HAI titers may not be the most appropriate method for assessing the immune response to intranasal vaccines, which is an issue that needs to be urgently addressed in Europe because of the likely application for licensure for a live ca
intranasal vaccine. The live ca
intranasal vaccine is as efficacious as the parenteral vaccine in preventing culture-positive influenza illness, but variable proportions of recipients (ranging from 10 to 32% for influenza B virus, 39 to 92% for influenza A H1N1 virus, and 28 to 86% for influenza A H3N2 virus) achieve reciprocal HAI titers of ≥32 (≥40 is one of the CHMP criteria for vaccine assessment) (5
). Thus, while many studies indicate that following immunization with inactivated virus vaccines, HAI antibody titers of approximately 1:30 to 1:40 represent the 50% protective level of antibody (28
), serum HAI responses to intranasally delivered vaccine correlate less well with protection.
It is generally considered that IgA is the main effector antibody of the mucosal immune system, whereas the origin and role of the observed IgG antibody in the nasal cavity are less certain. One possibility is that this IgG reaches the mucosal lumen by transudation from the circulation (39
), with studies of mice indicating that IgG antibodies are capable of providing protective mucosal immunity (12
). In this regard, it seems important to elicit a strong mucosal IgG response to vaccination in order to achieve optimal virus neutralization at the portal of virus entry. In our study, the parenterally administered vaccine elicited significantly better mucosal IgG antibody responses to each vaccine strain than the intranasal vaccines, which contrasts with its ability to elicit mucosal IgA responses. Some recipients of the intranasal vaccine containing 30 μg LTK63 and biovector developed mucosal IgG responses.
Immunity to influenza virus infection in humans is multifactorial, and the precise contributions of innate immunity, serum IgG to hemagglutinin and neuraminidase, local IgG and IgA, and Th1- and Th2-type immune responses have been difficult to ascertain. Observations indicate that live vaccine virus infection-induced and inactivated vaccine-induced immunity involve different arms of the immune system, with sufficient antibody in either serum or nasal secretions being capable of conferring resistance (7
). We and others have shown that the current parenteral influenza vaccines elicit strain-specific HAI humoral antibodies in most healthy individuals but that only a minority develop nasal IgA responses (5
). Parenterally administered vaccines are expensive and inconvenient to deliver, and the need for injections affects vaccine uptake (31
). In order to achieve better protection, new influenza vaccines should aim to induce both mucosal and systemic antibodies. The use of an intranasal vaccine containing LTK63 with biovector as a mucosal adjuvant and a special delivery system shows promise in this respect, but whether the immune response in humans is adequate to prevent or ameliorate influenza virus infection has not been demonstrated, and further studies are required to answer this question. Overall, both intranasal and intramuscular vaccines were easy to administer and well tolerated, although the incidence of transient local pain and the use of analgesics or antipyretics were greater after intramuscular vaccination than after intranasal vaccination, which is consistent with previous findings (13
). The safety of the intranasal vaccines containing the LTK63 enterotoxin mutant could not be evaluated effectively with the small numbers of subjects enrolled in this study. In 2002, the Swiss inactivated intranasal virosomal vaccine that contained E. coli
heat-labile holotoxin as an adjuvant was withdrawn from the market when postmarketing surveillance suggested a strong association between vaccination and Bell's palsy (24
). No serious adverse events were reported with the Swiss vaccine in prelicensure trials conducted among 1,218 volunteers during four winter seasons in 1996 to 1999. Subsequently, 107 case reports of Bell's palsy with vaccine exposure were identified in German-speaking parts of Switzerland, corresponding to 13 excess cases per 10,000 vaccinees (24
). Herpes simplex virus has been implicated in the pathogenesis of Bell's palsy, but the involvement of herpes simplex virus in the Swiss cases and the possible role of residual enterotoxin activity or local inflammatory responses to the holotoxin-containing vaccine are unknown. Cholera toxin binds to GM1 gangliosides expressed on epithelial cells and is able to enter the olfactory bulb via the olfactory epithelium after intranasal delivery, causing inflammatory responses in the central nervous system (38
). In contrast, the LTK63 mutant of the heat-labile enterotoxin from E. coli
appears to be safe and noninflammatory in the olfactory bulb in animal models (26
). Nevertheless, despite evidence for its safety in animals, reassurance that LTK63 does not cause neurological problems in humans will require large clinical trials, which should proceed cautiously.
The reemergence of highly pathogenic avian influenza H5N1 viruses in humans in 2004-2005 in Vietnam, Thailand, Cambodia, Indonesia, China, and, most recently, Turkey highlights the continuing pandemic threat posed by these viruses. In this study, the recipients of two trivalent intramuscular doses of vaccine, with each dose containing 15 μg A/Duck/Singapore/97 (H5N3) hemagglutinin, responded with a GM MN antibody titer of almost 1 in 30, a level comparable to that observed in our study of the monovalent H5N3 vaccine (25
). Our findings after one and two 15-μg doses of A/Duck/Singapore/97 (H5N3) vaccine suggest that the immune response to the H5N3 vaccine given in a multivalent preparation is no better or worse than that in a monovalent formulation, with the caveat that the serological tests were not concurrent and the studies were phase 1 evaluations with a small number of vaccinees. Recent WHO meetings have highlighted the need for improved influenza vaccines providing long-lasting, cross-subtype protection. The finding that mucosal delivery of an inactivated monovalent H3N2 vaccine that was administered with a mutant derivative of E. coli
heat-labile enterotoxin, LT(R192G) (which possesses reduced toxicity), can completely protect mice against lethal challenge with a highly pathogenic avian H5N1 virus isolated from humans suggests that a strategy of mucosal vaccination might stimulate cross-protection against multiple influenza virus subtypes, including those with pandemic potential (36
). We conclude that the vaccine containing the detoxified mutant LTK63 derivative of heat-labile enterotoxin from E. coli
represents a promising novel vaccine candidate that warrants further study.