Virus-specific and shared gene expression signatures in immune cells after vaccination in response to influenza and vaccinia stimulation

BackgroundIn the vaccine era, individuals receive multiple vaccines in their lifetime. Host gene expression in response to antigenic stimulation is usually virus-specific; however, identifying shared pathways of host response across a wide spectrum of vaccine pathogens can shed light on the molecular mechanisms/components which can be targeted for the development of broad/universal therapeutics and vaccines.MethodWe isolated PBMCs, monocytes, B cells, and CD8+ T cells from the peripheral blood of healthy donors, who received both seasonal influenza vaccine (within <1 year) and smallpox vaccine (within 1 - 4 years). Each of the purified cell populations was stimulated with either influenza virus or vaccinia virus. Differentially expressed genes (DEGs) relative to unstimulated controls were identified for each in vitro viral infection, as well as for both viral infections (shared DEGs). Pathway enrichment analysis was performed to associate identified DEGs with KEGG/biological pathways.ResultsWe identified 2,906, 3,888, 681, and 446 DEGs in PBMCs, monocytes, B cells, and CD8+ T cells, respectively, in response to influenza stimulation. Meanwhile, 97, 120, 20, and 10 DEGs were identified as gene signatures in PBMCs, monocytes, B cells, and CD8+ T cells, respectively, upon vaccinia stimulation. The majority of DEGs identified in PBMCs were also found in monocytes after either viral stimulation. Of the virus-specific DEGs, 55, 63, and 9 DEGs occurred in common in PBMCs, monocytes, and B cells, respectively, while no DEGs were shared in infected CD8+ T cells after influenza and vaccinia. Gene set enrichment analysis demonstrated that these shared DEGs were over-represented in innate signaling pathways, including cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor, Toll-like receptor signaling, RIG-I-like receptor signaling pathways, cytosolic DNA-sensing pathways, and natural killer cell mediated cytotoxicity.ConclusionOur results provide insights into virus-host interactions in different immune cells, as well as host defense mechanisms against viral stimulation. Our data also highlights the role of monocytes as a major cell population driving gene expression in ex vivo PBMCs in response to viral stimulation. The immune response signaling pathways identified in this study may provide specific targets for the development of novel virus-specific therapeutics and improved vaccines for vaccinia and influenza. Although influenza and vaccinia viruses have been selected in this study as pathogen models, this approach could be applicable to other pathogens

Development of a Novel Efficient Fluorescence-Based Plaque Reduction Microneutralization Assay for Measles Virus Immunity▿

The measurement of functional measles virus-specific neutralizing antibodies is of considerable interest for vaccine-related research. In this study, we developed and standardized a simple, rapid, highly sensitive, and reproducible fluorescence-based plaque reduction microneutralization (PRMN) assay with visual and automated readout, using a recombinant measles virus engineered to express enhanced green fluorescent protein. The assay is performed in micro format, requires less time to complete (2 versus 4 to 7 days), and is less labor-intensive and less costly than the classical plaque reduction neutralization (PRN) test, widely accepted as the “gold standard” in measles serology. Two available WHO international anti-measles virus standards and one in-house reference serum were used to develop and standardize the new assay. The mean PRMN values from repeated assays were found to be similar to those reported in the literature or assigned to the WHO standards by the classical PRN assay. For validation, we used three groups of low, moderate, and high measles virus vaccine responders’ sera with moderate values of correlation in antibody levels (mIU/ml) between PRMN and the Dade Behring immunoglobulin G enzyme immunoassay (EIA). The PRMN assay was more sensitive at low antibody levels and more informative in terms of protection than this commercial EIA. In conclusion, we have developed and validated a sensitive and high-throughput measles virus-specific PRMN that can be readily used in large population-based measles studies

Current perspectives in assessing humoral immunity after measles vaccination

Introduction: Repeated measles outbreaks in countries with relatively high vaccine coverage are mainly due to failure to vaccinate and importation; however, cases in immunized individuals exist raising questions about suboptimal measles vaccine-induced humoral immunity and/or waning immunity in a low measles-exposure environment. Areas covered: The plaque reduction neutralization measurement of functional measles-specific antibodies correlates with protection is the gold standard in measles serology, but it does not assess cellular-immune or other parameters that may be associated with durable and/or protective immunity after vaccination. Additional correlates of protection and long-term immunity and new determinants/signatures of vaccine responsiveness such as specific CD46 and IFI44L genetic variants associated with neutralizing antibody titers after measles vaccination are under investigation. Current and future systems biology studies, coupled with new technology/assays and analytical approaches, will lead to an increasingly sophisticated understanding of measles vaccine-induced humoral immunity and will identify ‘signatures’ of protective and durable immune responses. Expert opinion: This will translate into the development of highly predictive assays of measles vaccine efficacy, effectiveness, and durability for prospective identification of potential low/non-responders and susceptible individuals who require additional vaccine doses. Such new advances may drive insights into the development of new/improved vaccine formulations and delivery systems

Associations of adaptive immune cell subsets with measles, mumps, and Rubella−Specific immune response outcomes

The measles-mumps-rubella (MMR) vaccine has been widely used in the US, but measles and mumps outbreaks remain a public health issue in the US and elsewhere, even among individuals immunized with 2 doses of the vaccine. Immune correlates of vaccine-elicited protection against disease are typically assessed with serum antibody assays, but in some cases, these correlates fail to predict immunity, with the complexity and heterogeneity of the immune response. We used multicolor flow cytometry to evaluate changes in the frequency of peripheral T and B cell subsets in 82 study participants after receipt of a third dose of the M-M-RII vaccine (Merck & Co, Inc). We assessed correlations between flow cytometry variables and measles virus (MV), mumps virus (MuV), or rubella virus (RV)−specific immune response outcomes. Following a third vaccine dose, major changes were observed in the T-cell compartment. CD4+ T cell subsets were significantly increased from baseline to day 28, whereas CD8+ T cell subsets were predominantly decreased. Changes in regulatory T cells (Tregs) correlated with RV- and MV-specific immune outcomes and with high- and low−RV antibody responder groups, implicating the importance of Tregs in regulating MMR vaccine−induced immune responses. This information may help define additional correlates of protection and aid in the design of improved vaccines

Associations between single nucleotide polymorphisms in cellular viral receptors and attachment factor-related genes and humoral immunity to rubella vaccination.

Viral attachment and cell entry host factors are important for viral replication, pathogenesis, and the generation and sustenance of immune responses after infection and/or vaccination, and are plausible genetic regulators of vaccine-induced immunity.Using a tag-SNP approach in candidate gene study, we assessed the role of selected cell surface receptor genes, attachment factor-related genes, along with other immune genes in the genetic control of immune response variations after live rubella vaccination in two independent study cohorts.Our analysis revealed evidence for multiple associations between genetic variants in the PVR, PVRL2, CD209/DC-SIGN, RARB, MOG, IL6 and other immune function-related genes and rubella-specific neutralizing antibodies after vaccination (meta p-value <0.05).Our results indicate that multiple SNPs from genes involved in cell adhesion, viral attachment, and viral entry, as well as others in genes involved in signaling and/or immune response regulation, play a role in modulating humoral immune responses following live rubella vaccination

Profiling of Measles-Specific Humoral Immunity in Individuals Following Two Doses of MMR Vaccine Using Proteome Microarrays

Introduction: Comprehensive evaluation of measles-specific humoral immunity after vaccination is important for determining new and/or additional correlates of vaccine immunogenicity and efficacy. Methods: We used a novel proteome microarray technology and statistical modeling to identify factors and models associated with measles-specific functional protective immunity in 150 measles vaccine recipients representing the extremes of neutralizing antibody response after two vaccine doses. Results: Our findings demonstrate a high seroprevalence of antibodies directed to the measles virus (MV) phosphoprotein (P), nucleoprotein (N), as well as antibodies to the large polymerase (L) protein (fragment 1234 to 1900 AA). Antibodies to these proteins, in addition to anti-F antibodies (and, to a lesser extent, anti-H antibodies), were correlated with neutralizing antibody titer and/or were associated with and predictive of neutralizing antibody response. Conclusion: Our results identify antibodies to specific measles virus proteins and statistical models for monitoring and assessment of measles-specific functional protective immunity in vaccinated individuals