Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P=0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P=1.00 and P=0.33, respectively). The mucosal prime–parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime–parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.     

Immunization with recombinant Helicobacter pylori urease decreases colonization levels following experimental infection of rhesus monkeys

Rhesus monkeys, naturally colonized with H. pylori as indicated by culture and histology were immunized with either 40 mg recombinant H. pylori urease administered orally together with 25 microg Escherichia coli heat-labile enterotoxin (LT) or immunized with LT alone. An initial 6 doses were administered over an 8 week period. All five vaccinated monkeys had a greater than two-fold rise in urease-specific serum IgG and IgA level and urease-specific salivary IgA was induced in 3 of 5 vaccinated animals after 6 or 7 doses of vaccine. Vaccination had no measurable therapeutic effect on H. pylori colonization. H. pylori was eradicated from these monkeys with a course of antimicrobials plus omeprazole, a 7th vaccine dose was given (10 months after the 6th dose) and they were rechallenged with H. pylori. Necropsy was performed 23 weeks after rechallenge and H. pylori colonization was determined by histological examination of 12 individual gastric sites. A significant reduction in colonization (p < or = 0.0001; Friedman's analysis of variance) was found in the vaccinated animals. Histopathologic examination of necropsy tissues also revealed a trend towards reduced gastritis and epithelial alterations in the vaccinated group compared to animals receiving LT alone. This study provides the first evidence for effective vaccination of nonhuman primates against H. pylori, and preliminary evidence that a reduction in bacterial density attributable to immunization may lessen gastric inflammation.     

Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG,IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine

The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-γ and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs.     

Safety and immunogenicity of phoP/phoQ-deleted Salmonella typhi expressing Helicobacter pylori urease in adult volunteers.

Salmonella typhi Ty800, deleted for the Salmonella phoP/phoQ virulence regulon has been shown to be a safe and immunogenic single dose oral typhoid fever vaccine in volunteers. This promising vaccine strain was modified to constitutively express a heterologous protein of Gram negative bacterial origin, Helicobacter pylori urease subunits A and B, yielding S. typhi strain Ty1033. Seven volunteers received single oral doses of > or = 10(10) colony forming units of Ty1033; an eighth volunteer received two doses 3 months apart. Side effects were similar to those observed previously in volunteers who received the unmodified vector Ty800. All volunteers had strong mucosal immune responses to vaccination as measured by increases in IgA-secreting cells in peripheral blood directed against S. typhi antigens. Seven of eight volunteers had convincing seroconversion as measured by increases in serum IgG directed against S. typhi flagella and lipopolysaccharide antigens by ELISA. No volunteer had detectable mucosal or humoral immune responses to the urease antigen after immunization with single doses of Ty1033. A subset of three volunteers received an oral booster vaccination consisting of recombinant purified H. pylori urease A/B and E. coli heat labile toxin adjuvant 15 days after immunization with Ty1033. None of three had detectable humoral or mucosal immune responses to urease. Expression of a stable immunogenic protein in an appropriately attenuated S. typhi vector did not engender detectable mucosal or systemic antibody responses; additional work will be needed to define variables important for immunogenicity of heterologous antigens carried by live S. typhi vectors in humans.     

A Phase I safety and immunogenicity trial of UBI microparticulate monovalent HIV-1 MN oral peptide immunogen with parenteral boost in HIV-1 seronegative human subjects

Thirty-three HIV-seronegative adults were recruited into a Phase I safety and immunogenicity HIV-1 vaccine trial. The immunogens were as follows: a synthetic, monovalent, octameric HIV-1 MN V3 peptide in aluminum hydroxide (alum) adjuvant administered by intramuscular delivery; and a similar product encapsulated in biodegradable micro-spheres composed of co-polymers of lactic and glycolic acids, administered by the oral route. These were administered in three sequential oral doses, followed by a parenteral boost. No serious adverse experiences were observed. Oral administration of this vaccine, alone or in combination with parenteral boosting, resulted in no significant humoral, cellular, or mucosal immune responses.     

Oral immunization with HpaA affords therapeutic protective immunity against H. pylori that is reflected by specific mucosal immune responses

In the present study, we evaluated the capacity of Helicobacter pylori adhesin A (HpaA), a H. pylori specific colonization factor, to induce therapeutic protection against H. pylori infection in mice. We found that oral immunization of H. pylori infected mice with HpaA induced protection, i.e. significant reduction in bacterial load in the stomach. This was even more pronounced when a combination of HpaA and urease was used. The protection was strongly related to specific mucosal CD4+ T cell responses with a Th1 profile as well as to mucosal IgA responses locally in the stomach. These findings suggest that HpaA is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori.     

Booster vaccination with a fractional dose of an oral cholera vaccine induces comparable vaccine-specific antibody avidity as a full dose: A randomised clinical trial

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval.To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS).We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower.Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations.ISRCTN registry identifier 11806026.     

Lack of immune interference between inactivated polio vaccine and inactivated rotavirus vaccine co-administered by intramuscular injection in two animal species

A parenteral inactivated rotavirus vaccine (IRV) in development could address three problems with current live oral rotavirus vaccines (ORV): their lower efficacy in low and middle-income countries (LMICs), lingering concerns about their association with intussusception, and their requirement for a separate supply chain with large volume cold storage. Adding a new parenteral IRV to the current schedule of childhood immunizations would be more acceptable if it could be combined with another injectable vaccine such as inactivated polio vaccine (IPV). Current plans for polio eradication call for phasing out oral polio vaccine (OPV) and transitioning to IPV, initially in LMICs as a single dose booster after two doses of OPV and ultimately as a two dose schedule. Today in many LMICs, IPV is administered as a standalone vaccine, which involves a separate cold chain and is relatively costly. We therefore tested in two animal models formulations of IPV with IRV to determine whether co-administration might interfere with the immune response to each product and spare antigen dose for both vaccines. Our results demonstrate that IRV when adjuvanted with alum and administered alone or in combination with IPV did not impair the immune responses to either rotavirus or poliovirus serotypes 1, 2 and 3. Similarly, IPV when formulated and administered alone or together with IRV induced comparable levels of neutralizing antibody to poliovirus type 1, 2 and 3. Furthermore, comparable antibody titers were observed in animals vaccinated with low, middle or high dose of IPV or IRV in combination. This dose sparing and the lack of interference between IPV and IRV administered together represent another step to support the further development of this novel combination vaccine for children.     

Intra-epithelial vaccination with COPV L1 DNA by particle-mediated DNA delivery protects against mucosal challenge with infectious COPV in beagle dogs

Protection against viral challenge with canine oral papillomavirus (COPV) was achieved by immunisation via particle-mediated DNA delivery (PMDD) of a plasmid encoding the COPV L1 gene to cutaneous and oral mucosal sites in beagle dogs. The initial dose of approximately 9 microg of DNA was followed by two booster doses at 6 week intervals. A similar approach was used to vaccinate a control group of animals with plasmid DNA encoding the Hepatitis B virus S gene. Following challenge at the oral mucosa with COPV all animals vaccinated with the COPV L1 gene were protected against disease. However five of six animals in the control group developed COPV induced papillomas at the oral mucosa. Both cell-mediated lymphoproliferative and humoral antibody responses to the DNA vaccine were observed. Our data indicate that PMDD of plasmid DNA can protect against mucosal challenge with papillomavirus.     

Oral administration of live Shigella vaccine candidates in rhesus monkeys show no evidence of competition for colonization and immunogenicity between different serotypes

Live oral monovalent Shigella flexneri 2a vaccine candidates as well as bivalent formulations with Shigella sonnei were evaluated in a rhesus monkey model for colonization and immunogenicity. Freshly harvested suspensions of S. flexneri 2a vaccine candidates WRSf2G12 and WRSf2G15 as well as S. sonnei vaccine candidate WRSs3 were nasogastrically administered to groups of rhesus monkeys, Macaca mulatta, either in a monovalent form or when combined with each other. The animals were monitored daily for physical well-being, stools were subjected to quantitative colony immunoblot assays for bacterial excretion and blood and stools were evaluated for humoral and mucosal immune responses. No clinical symptoms were noted in any group of animals and the vaccine candidates were excreted robustly for 48–72 h without significant changes in either the magnitude or duration of excretion when given as a monovalent or as bivalent mixtures. Similarly, immunological interferences were not apparent in the magnitude of humoral and mucosal immune responses observed toward Shigella-specific antigens when monkeys were fed monovalent or bivalent formulations. These results predict that a multivalent live oral vaccine of more than one serotype can have a favorable outcome for protection against shigellosis.     

Dual route vaccination for plague with emergency use applications

Here, we report a dual-route vaccination approach for plague, able to induce a rapid response involving systemic and mucosal immunity, whilst also providing ease of use in those resource-poor settings most vulnerable to disease outbreaks. This novel vaccine (VypVaxDuo) comprises the recombinant F1 and V proteins in free association. VypVaxDuo has been designed for administration via a sub-cutaneous priming dose followed by a single oral booster dose and has been demonstrated to induce early onset immunity 14 days after the primary immunisation; full protective efficacy against live organism challenge was achieved in Balb/c mice exposed to 2 × 10⁴ median lethal doses of Yersinia pestis Co92, by the sub-cutaneous route at 25 days after the oral booster immunisation. This dual-route vaccination effectively induced serum IgG and serum and faecal IgA, specific for F1 and V, which constitute two key virulence factors in Y. pestis, and is therefore suitable for further development to prevent bubonic plague and for evaluation in models of pneumonic plague. This is an essential requirement for control of disease outbreaks in areas of the world endemic for plague and is supported further by the observed exceptional stability of the primary vaccine formulation in vialled form under thermostressed conditions (40 °C for 29 weeks, and 40 °C with 75% relative humidity for 6 weeks), meaning no cold chain for storage or distribution is needed.In clinical use, the injected priming dose would be administered on simple rehydration of the dry powder by means of a dual barrel syringe, with the subsequent single booster dose being provided in an enteric-coated capsule suitable for oral self-administration.     

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the na?ve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.     

Active immunity is seen as a reduction in the cell response to oral live vaccine

Oral immunization elicits a response of antibody-secreting cells (ASC) in the peripheral blood; these cells are believed to originate in the mucosa and hence give information on the mucosal immune response. We have shown earlier that oral booster immunization is followed by an elevated ASC response reflecting an immunologic memory. In the present study we show that a booster dose of a live bacterial vaccine given at a time of active mucosal immunity elicits a low ASC response only. This is probably because the multiplication of the live vaccine is inhibited in the gut, resulting in a low actual dose of the antigen. This situation may be an example of the protective immunity manifested when an orally immunized person encounters the pathogen in nature, and could be used to assess the protective immunity.     

Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-like-particles associated with immunostimulating complexes

The undesirable side effects and variable efficacy of some oral live rotavirus vaccines in infants have necessitated alternative vaccine approaches. We evaluated a recombinant RFVP2/WaVP6 rotavirus-like-particle (2/6VLP) oral vaccine, using an immunostimulating complex (ISCOM) matrix as adjuvant, in a gnotobiotic (Gn) pig model of human rotavirus (HRV) disease. The 2/6VLPs adhered to the ISCOM-matrix (2/6VLP-ISCOM ) and were antigenic, but they failed to induce protection. However, when combined with attenuated (Att) HRV for oral priming, the 2/6VLP-ISCOM vaccine was effective as a booster and induced partial protection against virulent Wa HRV. The 250 microg 2/6VLP dose was more effective than 100 microg. The highest mean numbers of IgA antibody secreting cells evaluated by ELISPOT in intestinal lymphoid tissues were in pigs receiving AttHRV+2/6VLP-ISCOM or three doses of AttHRV and were associated with the highest protection rates.     

Oral vaccination with liposome-encapsulated recombinant fusion peptide of urease B epitope and cholera toxin B subunit affords prophylactic and therapeutic effects against H. pylori infection in BALB/c mice

A new fusion peptide CtUBE of cholera toxin B subunit and Helicobacter pylori urease B subunit epitope was expressed in Escherichia coli. With this fusion peptide, an oral liposome vaccine against H. pylori infection was prepared and evaluated in BALB/c mice. Based on the results of urease tests, quantitation of culturable bacteria colonies in mice stomachs and histological identification of gastritis, the mice were protected significantly after intragastric vaccination with this CtUBE liposome vaccine, which increased the content levels of specific anti-urease serum IgG and mucosal IgA for both prophylactic and therapeutic vaccination protocols. These results showed that the fusion peptide CtUBE retained immunogenicity and could be used as antigen in the development of an oral vaccine against H. pylori infection.     

Mucosally-administered human-simian immunodeficiency virus DNA and fowlpoxvirus-based recombinant vaccines reduce acute phase viral replication in macaques following vaginal challenge with CCR5-tropic SHIVSF162P3

Further advances are required in understanding protection from AIDS by T cell immunity across mucosal sites of virus transmission. We analysed a set of multigenic HIV and SHIV DNA and Fowlpoxvirus (FPV) prime and boost vaccines for immunogenicity and protective efficacy in outbred pigtail macaques when delivered via mucosal surfaces (intranasally or intrarectally). Intranasally delivered DNA, even when adjuvanted and given as a fine droplet spray, was neither immunogenic nor protective in macaques. Some protection from acute infection with a pathogenic vaginal SHIVSF162P3 challenge was, however, observed with a regimen involving intramuscular DNA vaccine priming followed by either intranasally or intrarectally delivered rFPV boosting. Interestingly, animals boosted with rFPV vaccine via either of these mucosal routes had poor circulating T cell responses prior to challenge with SHIV compared to those boosted via the intramuscular route. Nevertheless, the mucosally-vaccinated animals generated equivalent anamnestic mucosal and systemic SHIV-specific CD4 and CD8 T cell responses following SHIV administration, with significant reduction in acute plasma viremia against this vaginal challenge. Our data suggest strategies for effective priming of partial immunity to mucosal HIV-1 exposure utilizing systemic prime and mucosal boost vaccination strategies.     

Oral immunization of mice with a multivalent therapeutic subunit vaccine protects against Helicobacter pylori infection

Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.     

Viral vectored granulocyte-macrophage colony stimulating factor inhibits vaccine protection in an SIV challenge model: protection correlates with neutralizing antibody

In a previous vaccine study, we reported significant and apparently sterilizing immunity to high-dose, mucosal, simian immunodeficiency virus (SIV) quasi-species challenge. The vaccine consisted of vectors based on vesicular stomatitis virus (VSV) expressing simian immunodeficiency virus (SIV) gag and env genes, a boost with propagating replicon particles expressing the same SIV genes, and a second boost with VSV-based vectors. Concurrent with that published study we had a parallel group of macaques given the same doses of vaccine vectors, but in addition, we included a third VSV vector expressing rhesus macaque GM-CSF in the priming immunization only. We report here that addition of the vector expressing GM-CSF did not enhance CD8 T cell or antibody responses to SIV antigens, and almost completely abolished the vaccine protection against high-dose mucosal challenge with SIV. Expression of GM-CSF may have limited vector replication excessively in the macaque model. Our results suggest caution in the use of GM-CSF as a vaccine adjuvant, especially when expressed by a viral vector. Combining vaccine group animals from this study and the previous study we found that there was a marginal but significant positive correlation between the neutralizing antibody to a neutralization resistant SIV Env and protection from infection.     

Efficient systemic and mucosal responses against the HIV-1 Tat protein by prime/boost vaccination using the lipopeptide MALP-2 as adjuvant

A major goal of HIV-1 vaccine development is the induction of mucosal immune responses able to stop or reduce viral infection directly at the portal of entry. We established a heterologous prime/boost vaccination protocol based on intradermal priming with the HIV-1 Tat protein and intranasal boosting with the Tat protein co-administered with the mucosal adjuvant MALP-2. Strong Tat-specific humoral responses were elicited in vaccinated mice at both systemic and mucosal levels. The cellular responses were characterized by a Th1 dominant helper pattern. The heterologous prime/boost regimen was also able to induce Tat-specific CTL, which were absent in animals receiving the homologous prime boost scheme. Thus, the heterologous prime/boost protocol was the only regimen able to evoke both CTL and sIgA responses. This suggests that a similar approach can be exploited to develop multi-component vaccines against HIV-1 infections able to induce both systemic and mucosal immune responses.     

Intestinal immunity induced by inactivated poliovirus vaccine

In order to detect any intestinal immunity against poliovirus infection induced by parenteral vaccination with inactivated poliovirus vaccine (IPV), experiments were conducted in monkeys (Macaca radiata). All animals were seronegative (antibody not detected at 1:2 dilution of serum) before investigation. Sixteen monkeys were vaccinated with three doses of IPV at monthly intervals. Groups of four vaccinated and two seronegative control animals were fed 100 median monkey infectious doses (100 MID50) of poliovirus type 1 (Mahoney strain) at 1, 4, 7 and 12 months after vaccination. While all control monkeys excreted poliovirus in the throat and faeces from day 2 to days 18-22, none of the vaccinated monkeys excreted virus. Thus, a high degree of intestinal immunity against infection was found. Although no evidence of infection was seen, antibody booster response occurred in most monkey fed virus 7 and 12 months after vaccination.