Regular articleHumoral, mucosal, and cellular immune responses to oral Norwalk virus-like particles in volunteers
Introduction
Noroviruses are enteric viruses of the Caliciviridae family that are the major cause of epidemic gastroenteritis in the United States [1]. Norwalk virus (NV) is one strain within this group. Recent studies using new diagnostic assays developed with recombinant NV (rNV) particles have shown that the epidemiologic significance of NV infections has been previously greatly underestimated. New antibody prevalence studies indicate NV infections occur in young children and adults in developed countries more frequently than previously reported [2], [3], [4]. Norovirus infections are a particular concern in elderly residents of nursing homes [5]. In addition, NV caused illness among U.S. troops in Kuwait and has caused significant debilitating illness during recent military maneuvers in Somalia and Afghanistan, on aircraft carriers, and on pleasure cruise ships [6], [7], [8]. The virus is spread by food and water and from person-to-person contact [9], [10]. The clinical manifestations of NV infections include sudden onset of vomiting and/or diarrhea after a 24- to 48-h incubation period; symptoms typically last 12–24 h [7].
The successful cloning, sequencing, and expression of the major capsid protein of NV in insect cells using baculovirus recombinants led to the discovery that the capsid protein folds spontaneously into virus-like particles (VLPs) that lack nucleic acid [11], [12]. Such rNV particles may be produced in high concentrations (20 mg of purified particles per 2 × 108 infected cells), and these particles have desirable properties for use as a subunit vaccine [23]. Specifically, the particles are (i) stable following lyophilization and when exposed to acid (pH 2.5), (ii) highly immunogenic when injected parenterally into animals, (iii) immunogenic when given orally [13] or intranasally [14] to mice in the absence of adjuvant, and (iv) safe and immunogenic when given orally to antibody-positive volunteers [15]. Recombinant NV particles could be used to immunize against NV and as a carrier to deliver other vaccine antigens.
Norwalk VLPs have previously been investigated as an oral immunogen in volunteers. Seropositive volunteers studied at Baylor College of Medicine were given two oral doses of 100 μg of Norwalk VLPs, 3 weeks apart [15]. Even with this low dosage, 3 (60%) of 5 volunteers developed a rise in serum antibody after the first dose. After 250 μg of VLP with bicarbonate buffer, 15 (100%) of 15 volunteers developed a four-fold rise in ELISA antibody to rNV protein when measured 2 weeks after immunization. The seroconversion rate was encouraging, but the maximal titers achieved did not approach those after experimental infection [15].
The purpose of this study was to determine the safety and immune responses to escalating dosages of Norwalk VLPs given by the oral route in seropositive adult volunteers (the seroprevalence of NV antibody in the United States is at least 90%) in an attempt to increase the serum antibody levels to titers closer to those observed after experimental Norwalk virus infection. The local mucosal antibody responses in the gastrointestinal and genital tracts and the cellular immune responses to VLP were also evaluated.
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Volunteers
Volunteers were adults 18–40 years of age recruited from the Baltimore community who gave informed, signed consent. They were counseled and trained so that they understood the rationale for the study as well as the risks, benefits, and procedures involved. To ensure comprehension of the study and to document that informed consent had been elicited, the volunteers had to pass a written examination before participation. Prospective volunteers were carefully screened to ensure that they were in
Safety
In the 3 days after receiving VLP vaccine, no volunteer at any of the three doses experienced fever, diarrhea, or vomiting. Three volunteers who received 250 or 500 μg, but no volunteer who received 2000 μg, reported mild cramps after vaccination. Nausea was reported by one vaccinee (2000 μg) and one placebo recipient; headache and malaise were reported more commonly among placebo recipients.
Immune responses
The ASC responses are shown in Fig. 1. All 30 recipients of VLP vaccine, regardless of dosage, had a
Discussion
It is estimated that every American has one episode of gastroenteritis each year [22], and young children and the elderly are at particularly high risk [5], [23]. The widespread incidence of norovirus infections in different epidemiologic settings and the impact of illness has led to interest in developing a vaccine. Vaccine development has been promoted by the observation that immunity develops after infection, but immunity is short-term and homologous virus-specific [24], [25]. Recombinant NV
Acknowledgements
The investigators are grateful for the invaluable contributions of the staffs of the Adult Clinical Studies Section, the Cellular Immunology and Flow Cytometry Section, and Applied Immunology Section of the Center for Vaccine Development and, in particular, acknowledge the invaluable contribution of the volunteers from our community. This study was supported by the Enteric Pathogens Research Unit, NIAID contract NO1-AI65299.
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