Enhancement of live vaccines by co-delivery of immune modulating proteins

Live vaccines are gradually replaced by protein-based vaccines given the latter's better safety. But live vaccines seem to be more efficient via stimulation of the innate immune system.

Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors

BackgroundVisceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice.MethodologyAnalysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice.ConclusionsTaken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice.

IntroductionVaricella‐zoster virus (VZV), a human alphaherpesvirus 3, elicits both chickenpox and shingles and/or postherpetic neuralgia. A live attenuated vaccine (LAV) and glycoprotein E (gE) subunit vaccine were developed to prevent VZV‐induced diseases. We recently reported that single‐strand RNA (ssRNA) based on the intergenic region of the internal ribosome entry site of cricket paralysis virus (CrPV) is an effective adjuvant for protein‐based and virus‐like particle‐based vaccines. Here, Chinese hamster ovary expression system and an LAV from Oka/SK strains.MethodsWe appraised the adjuvant effect of the same CrPV ssRNA encoding the gE gene formulated in the two vaccines using VZV‐primed C57BL/6 mice and guinea pigs. Humoral immunity and cell‐mediated immunity were assessed by enzyme‐linked immunosorbent assay (ELISA) and ELISPOT in gE subunit vaccine and by ELISA and fluorescent antibody to membrane antigen in LAV.ResultsThe gE subunit vaccine‐induced gE‐specific antibodies and CD4+ T‐cell responses (indicated by interferon‐γ [IFN‐γ] and interleukin‐2 secretion) in the ssRNA‐based adjuvant containing the VZV gE gene. Therefore, an ssRNA adjuvant combined with gE antigen can trigger the innate immune response and induce an adaptive immune response to ultimately activate humoral and cell‐mediated responses. VZV LAV could also induce VZV‐specific antibodies and IFN‐γ stimulated by LAV, whereas the effect of ssRNA as a vaccine adjuvant could not be confirmed. However, the ssRNA adjuvant increased VZV‐specific neutralizing antibody response.ConclusionsTaken together, these results highlight that the gE subunit vaccine and LAV developed in this study can be functional VZV vaccines, and ssRNAs appear to function better as adjuvants in a subunit vaccine than in an LAV.

Immunisation of cattle against Babesia bovis combining a multi-epitope modified vaccinia Ankara virus and a recombinant protein induce strong Th1 cell responses but fails to trigger neutralising antibodies required for protection

The kinetics of gene expression related to innate and adaptive immunity in the lung and spleen following Newcastle disease virus (NDV) infection in vaccinated broiler chickens employing different vaccination regimes.

Subunit vaccines are safer but less immunogenic than live-attenuated vaccines or whole cell inactivated vaccines. Adjuvants are used to enhance and modulate antigen (Ag) immunogenicity, aiming to induce a protective and long-lasting immune response. Several molecules and formulations have been studied for their adjuvanticity, but only seven have been approved to formulate human vaccines. Metallic nanoparticles (MeNPs), particularly those containing gold and iron oxides, are widely used in medicine for diagnosis and therapy and have been used as carriers for drugs and vaccines. However, little is known about the immune response elicited by MeNPs or about their importance in the development of new vaccines. There is evidence that these particles display adjuvant characteristics, promoting cell recruitment, antigen-presenting cell activation, cytokine production, and inducing a humoral immune response. This review focuses on the characteristics of MeNPs that could facilitate the induction of a cellular immune response, particularly T-helper 1 and T-helper 17, and their potential functions as adjuvants for subunit vaccines.

BackgroundHerpes zoster (HZ) develops in up to 50% of unvaccinated individuals who live to 85 years of age, accounting for more than 1 million cases of HZ annually in the United States. A live attenuated vaccine (LAV) for HZ is U.S. FDA approved for persons 50 years or older, though CDC Advisory Committee on Immunization Practices (ACIP) recommendations are only for persons beginning at age 60 years. LAV efficacy at preventing HZ is ~70% for persons 50–59 years of age, with lower efficacy in older adults, and it is efficacious in preventing post-herpetic neuralgia (PHN) beyond the HZ prevention. The efficacy of LAV after vaccination wanes over time. A new adjuvanted HZ subunit vaccine (SUV), administered as a two-dose series, has greater than 95% efficacy against HZ in persons 50–69 years of age. SUV efficacy remains greater than 90% in persons vaccinated at age 70 years and older, including the subgroup older than 80 years of age. Overall efficacy of SUV against PHN approaches 90%. The waning rate of efficacy after SUV vaccination is unknown.MethodsTo estimate the relative cost-effectiveness of SUV, LAV and no vaccination (NV) strategies, a Markov model was developed based on published trials and data on vaccine efficacy persistence, quality of life, resource utilization, costs and disease epidemiology. The perspective was U.S. societal, and the cycle length was one year with a lifelong time horizon. SUV efficacy was estimated for the base case to wane at the same rate as LAV, all persons were assumed to receive both doses of SUV, and the cost of SUV included both doses.ResultsFor individuals vaccinated at age 50 years the incremental cost-effectiveness ratio (ICER) for LAV vs. NV was $142,811 per quality-adjusted life-year (QALY); at age 60 years the ICER dropped to $59,482 per QALY. The cost-effectiveness ratio of SUV approached that of LAV when the SUV cost approached $500 for persons vaccinated at age 50 years and when the cost was $400 for those vaccinated at age 60 years. The SUV cost that would result in achieving an ICER target of $100,000 per QALY for SUV vaccination vs. NV at age 50 years was $316; at age 60 years the cost was $638.ConclusionVaccination at age 60 years with SUV was more cost-effective than LAV when SUV cost was ~$450 or less. Vaccination with SUV at age 50 years appeared to be cost-effective if SUV cost was ~$315 or less.Disclosures All authors: No reported disclosures.

SummaryBackgroundYellow fever (YF), a mosquito-borne acute viral haemorrhagic illness, is endemic to many tropical and subtropical areas of Africa and Central and South America. Vaccination remains the most effective prevention strategy; however, as repeated outbreaks have exhausted vaccine stockpiles, there is a need for improved YF vaccines to meet global demand. A live-attenuated YF vaccine candidate (referred to as vYF) cloned from a YF-17D vaccine (YF-VAX®) sub-strain, adapted for growth in Vero cells cultured in serum-free media, is in clinical development. We report the innate and adaptive immune responses and the transcriptome profile of selected genes induced by vYF.MethodsHealthy adults aged 18–60 years were randomised at a 1:1:1:1 ratio to receive one dose of vYF at 4, 5 or 6 Log CCID50 or YF-VAX (reference vaccine), administered subcutaneously in the upper arm (ClinicalTrials.gov identifier: NCT04142086). Blood/serum samples were obtained at scheduled time points through 180 days (D180) post-vaccination. The surrogate endpoints assessed were: serum cytokine/chemokine concentrations, measured by bead-based Multiplex assay; peripheral blood vYF-specific IgG and IgM memory B cell frequencies, measured by FluoroSpot assay; and expression of genes involved in the immune response to YF-17D vaccination by RT-qPCR.FindingsThere was no increase in any of the cytokine/chemokine concentrations assessed through D14 following vaccination with vYF or YF-VAX, except for a slight increase in IP-10 (CXCL10) levels. The gene expression profiles and kinetics following vaccination with vYF and YF-VAX were similar, inclusive of innate (antiviral responses [type-1 interferon, IFN signal transduction; interferon-stimulated genes], activated dendritic cells, viral sensing pattern recognition receptors) and adaptive (cell division in stimulated CD4+ T cells, B cell and antibody) immune signatures, which peaked at D7 and D14, respectively. Increases in vYF-specific IgG and IgM memory B cell frequencies at D28 and D180 were similar across the study groups.InterpretationvYF-induced strong innate and adaptive immune responses comparable to those induced by YF-VAX, with similar transcriptomic and kinetic profiles observed.Funding10.13039/100004339Sanofi.

Globally, it is estimated that respiratory syncytial virus (RSV) causes 33 million new episodes of acute lower respiratory tract infection (LRTI) in children <5 years of age and ≈120,000 deaths annually. In infants, RSV represents the leading cause of hospitalization worldwide and the second commonest cause of mortality in low- and middle-income countries.1,2 RSV also causes significant disease in immunocompromised hosts and the elderly and has been associated with the development of asthma.3 The increasingly recognized burden of RSV disease has made the development of a vaccine(s) a global health priority. The World Health Organization recently released a roadmap to facilitate the development and implementation of vaccines and monoclonal antibodies (mAbs) and estimated that RSV vaccination will be available in the next 5–10 years.4 This review summarizes the strategies and challenges associated with RSV vaccine development and the vaccine candidates undergoing clinical evaluation, with a focus on those geared toward the pediatric population. THE STRUCTURE OF RSV RSV has a negative sense nonsegmented RNA genome that encodes 11 proteins: 3 are nonstructural (NS1/NS2—that counteract interferon responses—and M2-2), and 8 are structural proteins. Of those 8 proteins, 5 are internal [N, P, M, M2-1, L]), and 3 are embedded in the virion membrane: the small hydrophobic (SH), fusion (F) and attachment (G) glycoproteins. RSV G and F carry antigenic determinants that elicit neutralizing antibodies. However, F is the preferred target for vaccine, mAb and antiviral development because it plays an essential role in host cell viral entry, is highly conserved within and among RSV A and B subtypes and because of its 6 antigenic sites that elicit the production of high-potency neutralizing antibodies (≥90% of neutralizing antibodies are directed against this protein).5 Most of the G protein is covered in glycans, leaving the central conserved domain available for neutralizing antibody binding. Except for this domain, G is not well conserved and it is recognized by few neutralizing antibodies, which has reduced enthusiasm for it as a vaccine target. Our understanding of the F protein in its 2 conformations, prefusion (pre-F) and postfusion (post-F), has revolutionized the field of RSV biology. Pre-F, the active form of F on the virion, is metastable and switches unpredictably to the stable post-F conformation that once it is folded cannot return to the pre-F form. Antibodies that bind to pre-F are more efficient at neutralizing RSV than those against post-F. As examples, antibodies against site ϕ, a pre-F-specific epitope, are 150 times more potent than palivizumab that binds to site-II, present in both F conformations, while antibodies against site I, exclusively present in post-F, show weak or no neutralization.5 In addition, non-neutralizing antibodies to F, G and also SH, may inhibit infection by complement-mediated neutralization or antibody-dependent cell-mediated cytotoxicity. Furthermore, all viral antigens have the potential to induce protection by T-cell-mediated immunity. CHALLENGES FOR RSV VACCINE DEVELOPMENT Despite the burden associated with RSV, and after 60 years of active research, there is no licensed vaccine due in part of our incomplete understanding of the pathogenesis of the disease. In general, primary RSV infections are more severe; however, reinfections are common throughout life as immunity is neither complete nor long-lasting. The ideal vaccine should induce a more durable and improved immune response than natural infection. Legacy of the Formalin-inactivated Vaccine RSV vaccine development has been hindered after the safety concerns of the first RSV vaccine that was developed in the 1960s. The formalin-inactivated-whole virus alum-precipitated vaccine, which recent evidence indicating that it was directed against post-F, was associated in naive infants, but not older children, with enhanced RSV disease (ERD) and 2 deaths upon subsequent exposure to natural RSV. The mechanisms of ERD are not well understood, but it appears that an excess of non-neutralizing antibodies coupled with a skewed T-helper 2 (Th2) immune response, and complement deposition in the lungs contributed to its development. This is a critical aspect that is being considered for the development of inactivated vaccines, and strategies to assess safety risks according to the different vaccine platforms in the infant population are required. Target Populations There are different age groups that will benefit from RSV vaccines, and these might require different approaches: young RSV-naive infants (<4–6 months), children >6 months and the elderly. Vaccination of older children (2–5 years of age) may also limit transmission, as older siblings frequently introduce RSV into the household. Infants <4–6 Months This age group has an immature/developing immune system characterized by low expression of interferon, abundance of regulatory T cells with tolerogenic reactivity and a limited B-cell repertoire because of inefficient generation of somatic hypermutations. All these factors are associated with a poor response to foreign antigens and the generation of high-affinity matured antibodies. In addition, the presence of maternal antibodies may interfere with vaccine immunogenicity. Young infants represent the main target population because the peak of severe RSV disease occurs in the first 2–3 months of life. This age group would likely benefit from maternal vaccination or neutralizing mAbs administered at birth. The main goal of maternal vaccination is to boost neutralizing RSV titers and thereby transplacental antibody transfer. However, the optimal timing for vaccination (2nd or 3rd trimester) and the durability of protection in the infant need to be defined. This coupled with the high prevalence of hypergammaglobulinemia in low- and middle-income countries, associated with HIV or malaria, which impairs transplacental antibody transfer, suggest the need for high maternal antibody titers to compete for transfer. Nevertheless, RSV antibody transfer through breast-feeding (IgG > IgA) may complement the maternal vaccination strategy.6 Vaccinating pregnant women could be questioned if it exclusively benefits the infant and not the mother. The limited data available in pregnant women are mostly derived from influenza surveillance studies with rates of RSV infection varying from 0.2% to 13%, which likely underestimates the real incidence of RSV during pregnancy. The concerns regarding adverse fetal outcomes are relatively low, because this would not be the first time the mother's immune system encounters RSV antigens and the safety profile of other vaccines used in pregnancy, such as tetanus, diptheria and acellular pertussis (Tdap) or influenza, is excellent. A number of RSV maternal vaccines are currently in clinical development (Table 1).TABLE 1.: Landscape of RSV Vaccines Undergoing Clinical TrialsOlder Infants and Children Based on the experience of the formalin-inactivated-RSV vaccine, within this age group those who are naive at the time of vaccination might be at risk of ERD with protein vaccines. This target population would likely benefit most from live-attenuated or vectored vaccines. The Elderly On the other side of the spectrum, the immunosenescence of adults >65 years of age and the presence of additional comorbidities may compromise vaccine responses and the ability to assess efficacy. This population might benefit most from adjuvanted vaccines. Clinical Endpoints The ideal vaccine should be able to prevent severe disease and limit transmission, but the lack of a standard definition of severe disease or precise markers to assess severity in infants has been a barrier for vaccine development. Clinical endpoints that define a successful vaccine might be different depending on the target population. Hospitalization and other endpoints that capture the inpatient/outpatient burden of the disease, such as a reduction in medically significant visits for RSV infection, should be considered.7 Developing composite endpoints that include a combination of viral (and possibly bacterial) factors, clinical parameters, and fast turn-around point of care biomarkers could help with patient classification and to standardize definitions.8 Also, long-term follow-up is recommended, as studies suggest that interventions reducing the acute burden of RSV disease may also impact the development of recurrent wheezing/asthma.9 Immune Correlates of Protection Serum neutralizing antibodies (IgG against pre-F > post-F and G) represent the main surrogate of protection, as shown by the effectiveness of immunoprophylaxis with anti-F mAb (palivizumab) in high risk infants. However, a standardized protective threshold has not been defined yet. Newer systems biology approaches are helping to define the optimal correlates of protection, which are complex and depend on multiple factors, rather than a single cutoff value in antibody assays, and will need to be adjusted to each target population. Other cocorrelates of protection may include, F-specific epitope antibodies, mucosal IgA, interferon responses, antibody-dependent cell-mediated cytotoxicity and cell-mediated immunity. In addition, a balanced Th1/Th2 immune response, indicated by a high IgG2a/IgG1 ratio, is desirable. Other Factors The lack of an ideal animal model has also slowed down RSV vaccine development. Human challenge models mostly reproduce upper but not LRTI, limiting the generalizability of the results or the ability to assess the impact of vaccines on disease severity. There are also gaps in RSV epidemiology with lack of accurate information defining the temporal and geographic patterns of RSV circulation in inpatients/outpatients, across different age groups or RSV-associated mortality. Implementing robust multiplex polymerase chain reaction-based surveillance platforms could help to assess the impact of interventions on the burden of RSV disease, to identify possible escape mutants, or the contribution of other respiratory viruses causing RSV-like illnesses. VACCINE STRATEGIES The most effective approach to protect young infants and children from severe RSV infection may be a combined strategy using passive and active immunization: either maternal vaccination with stabilized pre-F or virus-like particles containing the F protein or mAb against pre-F at birth; followed by pediatric active immunization with a live vaccine, either attenuated RSV or the pre-F protein expressed from a virus vector. There are 39 vaccines candidates under development (http://www.path.org); of those 19 are undergoing clinical trials (Table 1).10 Protein Vaccines Particle Based The recombinant adjuvanted RSV post-F nanoparticle vaccine is the most advanced vaccine in clinical development. Results from a phase-3 clinical trial that enrolled 4636 pregnant women on the third trimester demonstrated a decrease in RSV hospitalizations in the offspring; however, the study did not meet the primary endpoint defined as prevention of medically significant RSV LRTI. The potential approval of this vaccine is being evaluated. It also aims to target elderly individuals and children >6 months to 5 years of age. Subunit Vaccines These vaccines consist of purified, adjuvanted proteins and use stabilized pre-F as the main antigen with promising results. They are mainly directed at pregnant women or the elderly because of the risk of ERD in RSV-naive infants. Other subunit vaccines in clinical or preclinical stages are using SH or G as main vaccine antigens. Live Vaccines Vector Based There are 5 vector-based vaccines in clinical development. The first 4 use adenovirus as a vector, while the other uses a modified vaccinia Ankara virus. Two of them are intended for use in pediatric seronegative patients. All of these vaccines express RSV F (pre-F > post-F depending on the vaccine) and 2 of them also express other viral antigens (N, M2 or G proteins). Live-attenuated vaccines (LAVs) represent an attractive alternative for older infants and young children. LAVs are administered intranasally and are able to elicit broad innate, humoral and cellular responses and replicate in the respiratory tract despite the presence of maternal antibodies. Importantly, these vaccines have not been associated with ERD and are considered safer in infants. The use of reverse genetics has made possible to incorporate different mutations in the viral genome, making LAV sufficiently immunogenic and, except for rhinorrhea, not associated with adverse events. There are 6 intranasal LAVs undergoing phase-1 clinical trials; 4 are using attenuated RSV, one Sendai virus as a backbone expressing RSV F and the last one is a chimeric vaccine using bacille Calmette-Guerin (BCG). The BCG/RSV vaccine is the only LAV intended to be administered systemically (subdermal) and in newborns. MONOCLONAL ANTIBODIES mAbs are also being evaluated for the prevention of RSV LRTI in young infants. Of those, suptavumab (REGN-2222), that targeted the pre-F-specific site V, has been discontinued from the market after it failed to prevent serious RSV LRTI in premature infants (primary endpoint). During the study, RSV type B was the predominant circulating strain and developed escape mutations that conferred resistance to this mAb. MK-1654 is an extended half-life mAb currently undergoing phase-I clinical trials and it is directed against antigenic site-IV (present the pre-F and post-F forms). Nirsevimab (MEDI8897) is a highly potent human neutralizing IgG1Κ targeting the pre-F-specific antigenic site ϕ. It also has an extended half-life because of modifications in the FC region using YTE technology. MEDI8897 is entering phase-3 clinical trials with the intent to provide passive immunization for prevention of severe RSV LRTI to all infants (preterm and full term), using a fixed, once per season intramuscular dose. SUMMARY Over the past decade, there have been significant advances in our knowledge of RSV molecular and structural biology and in the understanding of the human immune response to RSV. Despite the barriers, there are several opportunities for RSV vaccine development to protect the most vulnerable populations. The increasing interest of academic, industry and international bodies, such as the World Health Organization or Bill & Melinda Gates Foundation, is helping to move the field forward, promoting the implementation of surveillance platforms and standardization of clinical definitions, assays and surrogate markers of protection.

Chronic hepatitis B virus (HBV) infection is a global public health issue. There are >250 million people chronically infected with HBV, and these chronic carriers are at high risk of developing end-stage liver diseases and hepatocellular carcinoma. Patients with chronic hepatitis B (CHB) usually acquire the virus perinatally, while most patients infected during adulthood develop acute hepatitis B (AHB), which usually results in viral clearance. HBV infection is noncytopathic, and liver injury is mostly contributed by host immune responses. The virus is stealthy, since the infection rarely induces type I interferon response in the early phase. In AHB, viral infection is detected and restrained by the innate immune response, which is followed by a strong and robust adaptive immune response and accompanied by viral clearance. In patients with CHB, both innate and adaptive immune responses are weak and thus rarely lead to viral clearance. Interferon α and nucleos(t)ide analogues are 2 classes of approved antiviral therapies. The former treatment activates nature killer (NK) cells and NK T cells, which partially enhances the innate immune response, while the later treatment suppresses viral replication by inhibiting reverse transcriptase, which may restore the HBV-specific adaptive immune response. However, single or combined treatment are still far from achieving seroclearance of HBV surface antigen. Although the treatment response is unsatisfactory in current clinical trials using several immunomodulators for boosting antiviral immunity, immunotherapy that is able to induce immune surveillance is still the most promising modality for HBV cure in the future.

Vaccine effectiveness is mainly determined by the mechanism mediating protection, emphasizing the importance of unraveling the protective mechanism for novel pneumococcal vaccine development. We previously demonstrated that the regulatory T cell (Treg) immune response has a protective effect against pneumococcal infection elicited by the live-attenuated pneumococcal vaccine SPY1. However, the mechanism underlying this protective effect remains unclear. In this study, a short synthetic peptide (P17) was used to downregulate Tregs during immunization and subsequent challenges in a mouse model. In immunized mice, increase in immune cytokines (IL-12p70, IL-4, IL-5, and IL-17A) induced by SPY1 were further upregulated by P17 treatment, whereas the decrease in the infection-associated inflammatory cytokine TNF-α by SPY1 was reversed. P17 also inhibited the increase in the immunosuppressive cytokine IL-10 and inflammatory mediator IL-6 in immunized mice. More severe pulmonary injuries and more dramatic inflammatory responses with worse survival in P17-treated immunized mice indicated the indispensable role of the Treg immune response in protection against pneumococcal infection by maintaining a balance among acquired immune responses stimulated by SPY1. Further studies revealed that the significant elevation of active transforming growth factor β (TGF-β)1 by SPY1 vaccination activated FOXP3, leading to increased frequencies of CD4+CD25+Foxp3+ T cells. Moreover, SPY1 vaccination elevated the levels of Smad2/3 and phosphor-Smad2/3 and downregulated the negative regulatory factor Smad7 in a time-dependent manner during pneumococcal infection, and these changes were reversed by P17 treatment. These results illustrate that SPY1-stimulated TGF-β1 induced the generation of SPY1-specific Tregs via the Smad2/3 signaling pathway. In addition, SPY1-specific Tregs may participate in protection via the enhanced expression of PD-1 and CTLA-4. The data presented here extend our understanding of how the SPY1-induced acquired Treg immune response contributes to protection elicited by live-attenuated vaccines and may be helpful for the evaluation of live vaccines and other mucosal vaccine candidates.

ABSTRACTImmune response is a highly coordinated cascade involving all the subsets of peripheral blood mononuclear cells (PBMCs). In this study, RNA sequencing (RNA-Seq) analysis of PBMC subsets was done to delineate the systems biology behind immune protection of the vaccine in sheep and goats. The PBMC subsets studied were CD4+, CD8+, CD14+, CD21+, and CD335+ cells from day 0 and day 5 of sheep and goats vaccinated with Sungri/96 peste des petits ruminants virus. Assessment of the immune response processes enriched by the differentially expressed genes (DEGs) in all the subsets suggested a strong dysregulation toward the development of early inflammatory microenvironment, which is very much required for differentiation of monocytes to macrophages, and activation as well as the migration of dendritic cells into the draining lymph nodes. The protein-protein interaction networks among the antiviral molecules (IFIT3, ISG15, MX1, MX2, RSAD2, ISG20, IFIT5, and IFIT1) and common DEGs across PBMC subsets in both species identified ISG15 to be a ubiquitous hub that helps in orchestrating antiviral host response against peste des petits ruminants virus (PPRV). IRF7 was found to be the key master regulator activated in most of the subsets in sheep and goats. Most of the pathways were found to be inactivated in B lymphocytes of both the species, indicating that 5 days postvaccination (dpv) is too early a time point for the B lymphocytes to react. The cell-mediated immune response and humoral immune response pathways were found more enriched in goats than in sheep. Although animals from both species survived the challenge, a contrast in pathway activation was observed in CD335+ cells.IMPORTANCE Peste des petits ruminants (PPR) by PPR virus (PPRV) is an World Organisation for Animal Health (OIE)-listed acute, contagious transboundary viral disease of small ruminants. The attenuated Sungri/96 PPRV vaccine used all over India against this PPR provides long-lasting robust innate and adaptive immune response. The early antiviral response was found mediated through type I interferon-independent interferon-stimulated gene (ISG) expression. However, systems biology behind this immune response is unknown. In this study, in vivo transcriptome profiling of PBMC subsets (CD4+, CD8+, CD14+, CD21+, and CD335+) in vaccinated goats and sheep (at 5 days postvaccination) was done to understand this systems biology. Though there are a few differences in the systems biology across cells (specially the NK cells) between sheep and goats, the coordinated response that is inclusive of all the cell subsets was found to be toward the induction of a strong innate immune response, which is needed for an appropriate adaptive immune response.

Porcine reproductive and respiratory syndrome (PRRS) is a devastating viral disease affecting swine production, health and welfare throughout the world. Vaccination has been considered as one of the most economic tools for PRRS control. A synergistic action of the innate and the adaptive immune system of host is essential for developing a durable protective immunity to vaccine antigen. The peripheral blood mononuclear cells (PBMCs) play central role in immune system and are able to display gene expression patterns characteristics for certain infection. Therefore, the current study aimed to investigate the global transcriptome profiles of PBMCs to characterize the innate and the adaptive immune response to PRRS Virus (PRRSV) vaccine in Pietrain pigs. We employed nine Affymetrix gene chip porcine gene 1.0 ST array for the transcriptome profiling of PBMCs collected from three female piglets at immediately before (D0), at one (D1) and 28 d (D28) post PRRSV vaccination given at 4 wk (D0) of their age. Two pairwise contrasts were tested to characterize transcriptome alterations associated with the innate immune response (D1 vs. D0) and the adaptive immune response (D28 vs. D0). Normalization and statistical analysis of microarray data was performed with the ‘oligo’ and ‘limma’ R/Bioconductor package. With FDR < 0.05 and log2 fold change ± 1.5 as cutoff criteria, 83 and 53 transcripts were found to be differentially expressed in PBMCs during innate and adaptive response, respectively. The microarray expression results were technically validated by qRT-PCR. The gene ontology (GO) terms such as viral life cycle, regulation of lymphocyte activation, cytokine activity and inflammatory response were enriched during the innate immune response. The GO terms enriched during adaptive response includes cytolysis, T cell mediated cytotoxicity, immunoglobulin production. Significant enrichment of cytokine-cytokine receptor interaction, signaling by interleukins, viral mRNA translation, IFN-γ pathway and AP-1 transcription factor network pathways was indicating the involvement of altered genes in the antiviral defense. Network analysis has detected four module were functionally involved with functional network of innate immune transcriptional response and five modules were detected for adaptive immune responses. The innate immune transcriptional network found to be regulated by LCK, STAT3, ATP5B, UBB and RSP17. While TGFβ, IL7R, RAD21, SP1 and GZMB are responsible for coordinating the adaptive immune transcriptional response to PRRSV vaccine in PBMCs. Further work is required to determine whether polymorphisms linked to these genes affect the immune response to PRRSV vaccine in pigs.

Evaluation of the protective immune response of an attenuated strain of Toxoplasma gondii with long-term passages on the Gecko cell line