Karina Ramirez

Neonatal Immunization with a Sindbis Virus-DNA Measles Vaccine Induces Adult-Like Neutralizing Antibodies and Cell-Mediated Immunity in the Presence of Maternal Antibodies

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150+ B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-γ production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.

Intranasal vaccination with an adjuvanted Norwalk virus-like particle vaccine elicits antigen-specific B memory responses in human adult volunteers.

Noroviruses are the most frequent cause of acute gastroenteritis in humans of all ages. No vaccines are currently available. An intranasally delivered Norwalk (NV) virus-like particle (VLP) vaccine was recently shown to be well tolerated, immunogenic and to protect against infection in Phase 1 studies. Here, we examined B memory (B(M)) responses in volunteers who received the highest dosage levels of the NV-VLP vaccine (50 μg and 100 μg). We measured the frequency of NV-specific IgG and IgA-secreting B(M) cells in peripheral blood and the level of antibodies produced by these cells in culture. All subjects immunized with 100 μg of the NV-VLP vaccine and 90% of those who received 50 μg had significant IgA or IgG B(M) responses. The B(M) cell frequencies correlated with serum antibody levels and mucosally-primed antibody-secreting cell responses. This is the first demonstration of dose-dependent, functional B(M) responses in humans immunized intranasally with a NV-VLP vaccine.

Heterologous Prime-Boost Strategy to Immunize Very Young Infants against Measles: Pre-clinical Studies in Rhesus Macaques

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the “window of vulnerability” (age 4–9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon‐based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle‐free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild‐type virus challenge. A proteosome‐measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime–boost strategy.

Mucosally delivered Salmonella Typhi expressing the Yersinia pestis F1 antigen elicits mucosal and systemic immunity early in life and primes the neonatal immune system for a vigorous anamnestic response to parenteral F1 boost

Neonates respond poorly to conventional vaccines. This has been attributed, in part, to the immaturity of neonatal dendritic cells that lack full capacity for Ag presentation and T cell stimulation. We engineered an attenuated Salmonella Typhi strain to express and export the F1 Ag of Y. pestis (S. Typhi(F1)) and investigated its immunogenicity early in life using a heterologous prime-boost regimen. Newborn mice primed intranasally with a single dose of S. Typhi(F1) elicited mucosal Ab- and IFN-γ-secreting cells 1 wk after immunization. They also developed a potent and fast anamnestic response to a subsequent parenteral boost with F1-alum, which surpassed those of mice primed and boosted with S. Typhi(F1) or F1-alum. Neonatal priming with S. Typhi(F1), as opposed to priming with F1-alum, resulted in a more balanced IgG2a/IgG1 profile, enhanced avidity maturation and stimulation of B memory cells, and strong Th1-type cell-mediated immunity. S. Typhi(F1) enhanced the activation and maturation of neonatal CD11c+ dendritic cells, shown by increased expression of CD80, CD86, CD40, and MHC-II cell surface markers and production of proinflammatory cytokines IL-12, TNF-α, IL-6, and MCP-1. S. Typhi(F1)-stimulated neonatal DC had improved capacity for Ag presentation and T cell stimulation in vitro and induced F1-specific CD4+ and CD8+ T cell responses when adoptively transferred to newborn mice. Mucosal immunization with S. Typhi expressing a foreign Ag effectively primes the neonatal immune system for potent, fast, and broader responses to a parenteral Ag boost. Such a strategy can prevent infectious diseases, including those considered biowarfare threats, early in life.

Sindbis Virus-Based Measles DNA Vaccines Protect Cotton Rats against Respiratory Measles: Relevance of Antibodies, Mucosal and Systemic Antibody-Secreting Cells, Memory B Cells, and Th1-Type Cytokines as Correlates of Immunity

ABSTRACT Measles remains an important cause of pediatric morbidity and mortality in developing countries, especially among infants who are too young to receive the current licensed live attenuated measles vaccine. We developed two Sindbis virus DNA vaccines encoding the measles virus hemagglutinin (pMSIN-H) and fusion proteins (pMSINH-FdU) and examined their immunogenicities and protective efficacies when administered alone or followed by the live measles virus vaccine in cotton rats. Neutralizing antibodies, mucosal and systemic antibody-secreting cells, memory B cells, and gamma interferon-secreting T cells developed after priming and increased after boosting. pMSIN-H priming conferred 100% protection against pulmonary measles, whereas pMSINH-FdU protected only in conjunction with the live measles virus vaccine boost.

Mucosal IgA Responses in Healthy Adult Volunteers following Intranasal Spray Delivery of a Live Attenuated Measles Vaccine

ABSTRACT Measles remains an important cause of morbidity and mortality among children in the developing world. The goal of this study was to examine measles virus-specific mucosal immune responses in healthy immune (n = 24; plaque reduction neutralization [PRN] titers of ≥200 mIU/ml) and nonimmune (n = 24) young adult volunteers who received the monovalent Moraten measles vaccine via intranasal (spray delivery) or subcutaneous immunization. Serum, oral fluid, and nasal wash samples were examined for measles virus-specific and total IgG and IgA on day 0 (prior to vaccination) and on days 14, 28, and 90 after vaccination. Nonimmune subjects vaccinated subcutaneously developed high levels of measles virus PRN, IgG, and IgA antibodies in serum, oral fluid, and nasal washes. Total IgG and secretory IgA (sIgA) titers were increased in nasal washes, and total IgG was increased in oral fluid specimens. There was a strong correlation between PRN and measles virus-specific IgG titers measured in serum, oral fluid, and nasal washes, whereas a weak correlation was found between PRN and measles virus-specific IgA titers. Notably, intranasal measles vaccination resulted in increased production of measles virus-specific sIgA in oral fluid and nasal washes in nonimmune individuals, without evidence of a systemic immune response. In contrast, no significant vaccine-induced responses were observed in immune subjects, regardless of the route of immunization. These results demonstrate that (i) intranasal measles immunization can elicit a mucosal response independent of the induction of serum antibodies and (ii) both mucosal and systemic antibody responses following nasal or subcutaneous immunization are blunted by preexisting measles immunity.

Preclinical safety and biodistribution of Sindbis virus measles DNA vaccines administered as a single dose or followed by live attenuated measles vaccine in a heterologous prime-boost regimen.

Measles still causes considerable morbidity and mortality among infants and young children in developing countries. To develop a new public health tool to reduce this burden, we designed two Sindbis virus replicon vaccines encoding measles virus (MV) hemagglutinin (H) and fusion (F) proteins (pMSIN-H and pMSINHFdU). Our goal is to administer the vaccines to young infants at 6 and 10 weeks of age to prime the immune system to safely and effectively respond to subsequent immunization at age approximately 14 weeks with the licensed attenuated measles vaccine. In preparation for a phase 1 clinical trial, studies of plasmid distribution, integration, and toxicology were performed in rabbits. Biodistribution was assessed after a single DNA immunization delivered intradermally by needle-free injection. Toxicity was assessed using a heterologous prime-boost regimen consisting of a repeat-dose DNA prime followed by a live-attenuated measles vaccine boost. The only vaccine-related adverse effects observed were minimal transient erythema, edema, and inflammation confined to the injection site. Plasmids were detected in the subcutis and muscle at the site of inoculation. A small proportion of animals exhibited plasmids in the regional lymph nodes. There was no evidence of plasmid integration into the host genome. Both Sindbis-based vaccine plasmids were immunogenic in rabbits; pMSIN-H elicited higher virus-neutralizing antibody levels. Both vaccines were shown to be well tolerated and suitable for clinical trials and they are currently being tested in phase 1 studies in young adults.

Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies

The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-gamma-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.

Characterization of systemic and pneumonic murine models of plague infection using a conditionally virulent strain.

Yersinia pestis causes bubonic and pneumonic plague in humans. The pneumonic infection is the most severe and invariably fatal if untreated. Because of its high virulence, ease of delivery and precedent of use in warfare, Y. pestis is considered as a potential bioterror agent. No licensed plague vaccine is currently available in the US. Laboratory research with virulent strains requires appropriate biocontainment (i.e., Biosafety Level 3 (BSL-3) for procedures that generate aerosol/droplets) and secure facilities that comply with federal select agent regulations. To assist in the identification of promising vaccine candidates during the early phases of development, we characterized mouse models of systemic and pneumonic plague infection using the Y. pestis strain EV76, an attenuated human vaccine strain that can be rendered virulent in mice under in vivo iron supplementation. Mice inoculated intranasally or intravenously with Y. pestis EV76 in the presence of iron developed a systemic and pneumonic plague infection that resulted in disease and lethality. Bacteria replicated and severely compromised the spleen, liver and lungs. Susceptibility was age dependent, with younger mice being more vulnerable to pneumonic infection. We used these models of infection to assess the protective capacity of newly developed Salmonella-based plague vaccines. The protective outcome varied depending on the route and dose of infection. Protection was associated with the induction of specific immunological effectors in systemic/mucosal compartments. The models of infection described could serve as safe and practical tools for identifying promising vaccine candidates that warrant further potency evaluation using fully virulent strains in BSL-3 settings.