We examined the roles of cell- and antibody-mediated immunity in urease vaccine–induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [β2-microglobulin (−/−)] but not MHC class II knockout mice [I-Ab (−/−)]. In B cell knockout mice [μMT (−/−)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4+ cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II–restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.

Helicobacter pylori, a gram-negative spiral bacterium, is the cause of chronic superficial (type B) gastritis and peptic ulcer disease. The urease enzyme of H. pylori was expressed as an inactive recombinant protein in Escherichia coli, purified as particulate structures of 550-600 kDa molecular mass with a diameter of approximately 12 nm. Given orally, 5 micrograms of urease with an appropriate mucosal adjuvant, such as the labile toxin of E. coli, protected 60%-100% of mice against challenge with virulent Helicobacter felis. Protection correlated with the level of secretory IgA antibodies against urease. Oral administration of antigen was as effective or better than intragastric administration. Parenteral injection of antigen or intragastric administration of high-dose antigen without adjuvant elicited serum IgG but no IgA antibodies and did not confer protection. Recombinant urease as an oral vaccine candidate deserves further investigation as an approach to the prevention of Helicobacter-induced chronic gastroduodenal diseases in humans.

One of the challenges of developing influenza A vaccines is the diversity of antigenically distinct isolates. Previously, a novel hemagglutinin (HA) for H5N1 influenza was derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA elicited a broad antibody response against H5N1 isolates from different clades. We now report the development and characterization of a COBRA-based vaccine for both seasonal and pandemic H1N1 influenza virus isolates. Nine prototype H1N1 COBRA HA proteins were developed and tested in mice using a virus-like particle (VLP) format for the elicitation of broadly reactive, functional antibody responses and protection against viral challenge. These candidates were designed to recognize H1N1 viruses isolated within the last 30 years. In addition, several COBRA candidates were designed based on sequences of H1N1 viruses spanning the past 100 years, including modern pandemic H1N1 isolates. Four of the 9 H1N1 COBRA HA proteins (X1, X3, X6, and P1) had the broadest hemagglutination inhibition (HAI) activity against a panel of 17 H1N1 viruses. These vaccines were used in cocktails or prime-boost combinations. The most effective regimens that both elicited the broadest HAI response and protected mice against a pandemic H1N1 challenge were vaccines that contained the P1 COBRA VLP and either the X3 or X6 COBRA VLP vaccine. These mice had little or no detectable viral replication, comparable to that observed with a matched licensed vaccine. This is the first report describing a COBRA-based HA vaccine strategy that elicits a universal, broadly reactive, protective response against seasonal and pandemic H1N1 isolates. IMPORTANCEUniversal influenza vaccine approaches have the potential to be paradigm shifting for the influenza vaccine field, with the goal of replacing the current standard of care with broadly cross-protective vaccines. We have used COBRA technology to develop an HA head-based strategy that elicits antibodies against many H1 strains that have undergone genetic drift and has potential as a "subtype universal" vaccine. Nine HA COBRA candidates were developed, and these vaccines were used alone, in cocktails or in prime-boost combinations. The most effective regimens elicited the broadest hemagglutination inhibition (HAI) response against a panel of H1N1 viruses isolated over the past 100 years. This is the first report describing a COBRA-based HA vaccine strategy that elicits a broadly reactive response against seasonal and pandemic H1N1 isolates. Influenza vaccine efficacy is constantly undermined by antigenic variation in the circulating viral strains, particularly in the hemagglutinin (HA) and neuraminidase (NA) proteins. Current influenza vaccination strategies rely on changing the HA and NA components of the annual human influenza vaccine to ensure that they antigenically match circulating influenza strains (1, 2). Developing an influenza vaccine that is capable of providing broad and long-lasting protective antibody responses r...

A polymerase chain reaction (PCR) for the specific detection of Helicobacter pylori was developed with a single primer pair derived from the nucleotide sequence of the urease A gene of H. pylori. We achieved specific amplification of a 411-bp DNA fragment in H. pylori. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis and contained conserved single HinfI and AluI restriction sites. This fragment was amplified in all 50 strains of H. pylori tested, but it was not detected in other bacterial species, showing the PCR assay to be 100% specific. PCR DNA amplification was able to detect as few as 10 H. pylori cells. PCR detected H. pylori in 15 of 23 clinical human gastric biopsy samples, whereas culturing and microscopy detected H. pylori in only 7 of the samples found to be positive by PCR. Additional primer pairs based on the urease genes enabled the detection of H. pylori in paraffin-embedded human gastric biopsy samples. The detection of H. pylori by PCR will enable both retrospective and prospective analyses of clinical samples, elucidating the role of this organism in gastroduodenal disease.