Rajeev Rudraraju

Sendai virus-based RSV vaccine protects African green monkeys from RSV infection

Respiratory syncytial virus (RSV) is a serious disease of children, responsible for an estimated 160,000 deaths per year worldwide. Despite the ongoing need for global prevention of RSV and decades of research, there remains no licensed vaccine. Sendai virus (SeV) is a mouse parainfluenza virus-type 1 which has been previously shown to confer protection against its human cousin, human parainfluenza virus-type 1 in African green monkeys (AGM). Here is described the study of a RSV vaccine (SeVRSV), produced by reverse genetics technology using SeV as a backbone to carry the full-length gene for RSV F. To test for immunogenicity, efficacy and safety, the vaccine was administered to AGM by intratracheal (i.t.) and intranasal (i.n.) routes. Control animals received the empty SeV vector or PBS. There were no booster immunizations. SeV and SeVRSV were cleared from the URT and LRT of vaccinated animals by day 10. Antibodies with specificities toward SeV and RSV were detected in SeVRSV primed animals as early as day ten after immunizations in both sera and nasal wash samples. One month after immunization all test and control AGM received an i.n. challenge with RSV-A2. SeVRSV-vaccinated animals exhibited reduced RSV in the URT compared to controls, and complete protection against RSV in the LRT. There were no clinically relevant adverse events associated with vaccination either before or after challenge. These data encourage advanced testing of the SeVRSV vaccine candidate in clinical trials for protection against RSV.

Respiratory Syncytial Virus: Current Progress in Vaccine Development

Respiratory syncytial virus (RSV) is the etiological agent for a serious lower respiratory tract disease responsible for close to 200,000 annual deaths worldwide. The first infection is generally most severe, while re-infections usually associate with a milder disease. This observation and the finding that re-infection risks are inversely associated with neutralizing antibody titers suggest that immune responses generated toward a first RSV exposure can significantly reduce morbidity and mortality throughout life. For more than half a century, researchers have endeavored to design a vaccine for RSV that can mimic or improve upon natural protective immunity without adverse events. The virus is herein described together with the hurdles that must be overcome to develop a vaccine and some current vaccine development approaches.

Phenotypes and functions of persistent Sendai virus-induced antibody forming cells and CD8+ T cells in diffuse nasal-associated lymphoid tissue typify lymphocyte responses of the gut

Lymphocytes of the diffuse nasal-associated lymphoid tissue (d-NALT) are uniquely positioned to tackle respiratory pathogens at their point-of-entry, yet are rarely examined after intranasal (i.n.) vaccinations or infections. Here we evaluate an i.n. inoculation with Sendai virus (SeV) for elicitation of virus-specific antibody forming cells (AFCs) and CD8(+) T cells in the d-NALT. Virus-specific AFCs and CD8(+) T cells each appeared by day 7 after SeV inoculation and persisted for 8 months, explaining the long-sustained protection against respiratory virus challenge conferred by this vaccine. AFCs produced IgM, IgG1, IgG2a, IgG2b and IgA, while CD8+ T cells were cytolytic and produced low levels of cytokines. Phenotypic analyses of virus-specific T cells revealed striking similarities with pathogen-specific immune responses in the intestine, highlighting some key features of adaptive immunity at a mucosal site.

Vitamin A Deficiency Disrupts Vaccine-Induced Antibody-Forming Cells and the Balance of IgA/IgG Isotypes in the Upper and Lower Respiratory Tract

Vaccination by intranasal (IN) inoculation with a replication-competent virus forms the basis of licensed and novel candidate respiratory viral vaccines (e.g., the cold-adapted influenza virus vaccine). A positive global impact of vaccination depends on vaccine efficacy in developing countries where dietary deficiencies are commonplace. The current study was designed using Sendai virus (SeV) as a model respiratory viral vaccine to test antibody-forming cell (AFC) residence and isotype expression in the context of a vitamin A deficiency (VAD). Samples were taken 1 mo after vaccination when AFCs generally reach their peak in healthy animals. In control animals on a healthy diet, SeV induced an antibody response with a relative bias toward IgA in the upper respiratory tract (URT, as sampled by nasal wash), and IgG in the lower respiratory tract (LRT, as sampled by bronchoalveolar lavage [BAL]). In the context of VAD, the SeV-specific IgA antibodies in the nasal wash were significantly reduced in favor of enhanced IgG antibodies in the BAL. When AFCs were examined in diffuse nasal-associated lymphoid tissues (d-NALT), lungs, cervical lymph nodes (CLN), and mediastinal lymph nodes (MLN), a similar pattern emerged. AFCs were most frequent in the d-NALT and most expressed IgA in control mice. In the context of VAD, these IgA-producing AFCs were significantly reduced in number, skewing the natural balance of IgA and IgG. Taken together, the results show that the VAD diet, which is well known for its association with immune defects in the gut, significantly alters AFC induction and isotype expression in the respiratory tract.

Respiratory tract epithelial cells express retinaldehyde dehydrogenase ALDH1A and enhance IgA production by stimulated B cells in the presence of vitamin A.

Morbidity and mortality due to viral infections are major health concerns, particularly when individuals are vitamin A deficient. Vitamin A deficiency significantly impairs mucosal IgA, a first line of defense against virus at its point of entry. Previous reports have suggested that CD11cHi dendritic cells (DCs) of the gastrointestinal tract produce retinaldehyde dehydrogenase (ALDH1A), which metabolizes vitamin A precursors to retinoic acid to support normal mucosal immunity. Given that the upper respiratory tract (URT) and gastrointestinal tract share numerous characteristics, we asked if the CD11cHi DCs of the URT might also express ALDH1A. To address this question, we examined both CD11cHi test cells and CD11cLo/neg control cells from nasal tissue. Surprisingly, the CD11cLo/neg cells expressed more ALDH1A mRNA per cell than did the CD11cHi cells. Further evaluation of CD11cLo/neg populations by PCR and staining of respiratory tract sections revealed that epithelial cells were robust producers of both ALDH1A mRNA and protein. Moreover, CD11cLo/neg cells from nasal tissue (and a homogeneous respiratory tract epithelial cell line) enhanced IgA production by lipopolysaccharide (LPS)-stimulated splenocyte cultures in the presence of the retinoic acid precursor retinol. Within co-cultures, there was increased expression of MCP-1, IL-6, and GM-CSF, the latter two of which were necessary for IgA upregulation. All three cytokines/chemokines were expressed by the LPS-stimulated respiratory tract epithelial cell line in the absence of splenocytes. These data demonstrate the autonomous potential of respiratory tract epithelial cells to support vitamin A-mediated IgA production, and encourage the clinical testing of intranasal vitamin A supplements in vitamin A deficient populations to improve mucosal immune responses toward respiratory tract pathogens and vaccines.

Reduced frequencies and heightened CD103 expression among virus-induced CD8+ T cells in the respiratory tract airways of Vitamin A deficient mice

ABSTRACT Vitamin A deficiency (VAD) has profound effects on immune responses in the gut, but its effect on other mucosal responses is less well understood. Sendai virus (SeV) is a candidate human parainfluenza virus type 1 (hPIV-1) vaccine and a candidate vaccine vector for other respiratory viruses. A single intranasal dose of SeV elicits a protective immune response against hPIV-1 within days after vaccination. To define the effect of VAD on acute responses toward SeV, we monitored both antibodies and CD8+ T cells in mice. On day 10 following SeV infection, there was a trend toward lower antibody activities in the nasal washes of VAD mice than in those of controls, while bronchoalveolar lavage (BAL) fluid and serum antibodies were not reduced. In contrast, there was a dramatic reduction of immunodominant CD8+ T cell frequencies in the lower respiratory tract (LRT) airways of VAD animals. These T cells also showed unusually high CD103 (the αE subunit of αEβ7) expression patterns. In both VAD and control mice, E-cadherin (the ligand for αEβ7) was better expressed among epithelial cells lining the upper respiratory tract (URT) than in LRT airways. The results support a working hypothesis that the high CD103 expression among T cell populations in VAD mice alters mechanisms of T cell cross talk with URT and LRT epithelial cells, thereby inhibiting T cell migration and egress into the lower airway. Our data emphasize that the consequences of VAD are not limited to gut-resident cells and characterize VAD influences on an immune response to a respiratory virus vaccine.

Respiratory Syncytial Virus Human Experimental Infection Model: Provenance, Production, and Sequence of Low-Passaged Memphis-37 Challenge Virus

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children and is responsible for as many as 199,000 childhood deaths annually worldwide. To support the development of viral therapeutics and vaccines for RSV, a human adult experimental infection model has been established. In this report, we describe the provenance and sequence of RSV Memphis-37, the low-passage clinical isolate used for the models reproducible, safe, experimental infections of healthy, adult volunteers. The predicted amino acid sequences for major proteins of Memphis-37 are compared to nine other RSV A and B amino acid sequences to examine sites of vaccine, therapeutic, and pathophysiologic interest. Human T- cell epitope sequences previously defined by in vitro studies were observed to be closely matched between Memphis-37 and the laboratory strain RSV A2. Memphis-37 sequences provide baseline data with which to assess: (i) virus heterogeneity that may be evident following virus infection/transmission, (ii) the efficacy of candidate RSV vaccines and therapeutics in the experimental infection model, and (iii) the potential emergence of escape mutants as a consequence of experimental drug treatments. Memphis-37 is a valuable tool for pre-clinical research, and to expedite the clinical development of vaccines, therapeutic immunomodulatory agents, and other antiviral drug strategies for the protection of vulnerable populations against RSV disease.

Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal influenza virus vaccine in vitamin A deficient mice.

Vitamin A deficiency (VAD) is a leading cause of pediatric morbidity and mortality due to infectious diseases. Recent pre-clinical studies have revealed that VAD impairs mucosal IgA-producing antibody forming cell (AFC) responses toward a paramyxovirus vaccine in the upper respiratory tract (URT), thus impeding a first line of defense at the pathogens point-of-entry. The studies described here tested the hypothesis that VAD may also impair immune responses after FluMist vaccinations. Results show that (i) IgA-producing antibody forming cells (AFCs) are significantly reduced following FluMist vaccination in VAD mice, and (ii) oral doses of either retinyl palmitate or retinoic acid administered on days 0, 3, and 7 relative to vaccination rescue the response. Data encourage the conduct of clinical studies to determine if there are FluMist vaccine weaknesses in human VAD populations and to test corrective supplementation strategies. Improvements in vaccine efficacy may ultimately reduce the morbidity and mortality caused by influenza virus worldwide.

Intranasal Administration of Retinyl Palmitate with a Respiratory Virus Vaccine Corrects Impaired Mucosal IgA Response in the Vitamin A-Deficient Host

ABSTRACT Our previous studies showed that intranasal vaccination of vitamin A-deficient (VAD) mice failed to induce normal levels of upper respiratory tract IgA, a first line of defense against respiratory virus infection. Here we demonstrate that the impaired responses in VAD animals are corrected by a single intranasal application of retinyl palmitate with the vaccine. Results encourage the clinical testing of intranasal vitamin A supplements to improve protection against respiratory viral disease in VAD populations.

Clonally Related CD8+ T Cells Responsible for Rapid Population of Both Diffuse Nasal-Associated Lymphoid Tissue and Lung After Respiratory Virus Infection

The immune system has evolved to use sophisticated mechanisms to recruit lymphocytes to sites of pathogen exposure. Trafficking pathways are precise. For example, lymphocytes that are primed by gut pathogens can, in some cases, be imprinted with CCR9 membrane receptors, which can influence migration to the small intestine. Currently, little is known about T cell trafficking to the upper respiratory tract or the relationship between effectors that migrate to the diffuse nasal-associated lymphoid tissue (d-NALT), the lower airways, and the lung. To determine whether a T cell primed by Ag from a respiratory pathogen is imprinted for exclusive trafficking to the upper or lower respiratory tract or whether descendents from that cell have the capacity to migrate to both sites, we inoculated mice by the intranasal route with Sendai virus and conducted single-cell–sequencing analyses of CD8+ T lymphocytes responsive to a Kb-restricted immunodominant peptide, FAPGNYPAL (Tet+). Cells from the d-NALT, lung airways (bronchoalveolar lavage), lung, and mediastinal lymph node were examined 10 d postinfection to determine TCR usage and clonal relationships. We discovered that 1) Tet+ cells were heterogeneous but preferentially used TCR elements TRAV6, TRAV16, and TRBD1; 2) both N and C termini of Vα and Vβ TCR junctions frequently encompassed charged residues, perhaps facilitating TCR αβ pairing and interactions with a neutral target peptide; and 3) T cells in the d-NALT were often clonally related to cells in the lower respiratory tract.