Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

Intranasal Nanoparticle Vaccination Elicits a Persistent, Polyfunctional CD4 T Cell Response in the Murine Lung Specific for a Highly Conserved Influenza Virus Antigen That Is Sufficient To Mediate Protection from Influenza Virus Challenge.

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

Cutting Edge: Antitumor Immunity by Pathogen-Specific CD8 T Cells in the Absence of Cognate Antigen Recognition.

Cancer prognosis often correlates with the number of tumor-infiltrating CD8 T cells, but many of these cells recognize pathogens that commonly infect humans. The contribution of pathogen-specific "bystander" CD8 T cells to antitumor immunity remains largely unknown. Inflammatory cytokines are sufficient for memory CD8 T cell activation and gain of effector functions, indicating tumor-derived inflammation could facilitate pathogen-specific CD8 T cells to participate in tumor control. In this study, we show in contrast to tumor-specific CD8 T cells that pathogen-specific primary memory CD8 T cells inside tumor were not able to exert their effector functions and influence tumor progression. However, infection-induced memory CD8 T cells with defined history of repeated Ag encounters (i.e., quaternary memory) showed increased sensitivity to tumor-derived inflammation that resulted in activation, gain of effector functions, and better control of tumor growth. Thus, memory CD8 T cells with heightened ability to recognize environmental inflammatory stimuli can contribute to antitumor immunity in the absence of cognate Ag recognition.

Neutralization of rhesus cytomegalovirus IL-10 reduces horizontal transmission and alters long-term immunity.

Human cytomegalovirus (HCMV) causes severe disease in infants and immunocompromised people. There is no approved HCMV vaccine, and vaccine development strategies are complicated by evidence of both persistent infection and reinfection of people with prior immunity. The greatest emphasis has been placed on reducing transmission to seronegative pregnant women to prevent vertical transmission and its potentially severe sequelae. Increasing evidence suggests that the earliest host-HCMV interactions establish conditions for viral persistence, including evasion of host immune responses to the virus. Using a nonhuman primate model of HCMV infection, we show that rhesus macaques immunized against viral interleukin-10 (IL-10) manifest delayed rhesus cytomegalovirus (RhCMV) acquisition and altered immune responses to the infection when it does occur. Among animals with the greatest antiviral IL-10-neutralizing activity, the timing of RhCMV seroconversion was delayed by an average of 12 weeks. After acquisition, such animals displayed an antibody response to the new infection, which peaked as expected after 2 weeks but then declined rapidly. In contrast, surprisingly, vaccination with glycoprotein B (gB) protein had no discernible impact on these outcomes. Our results demonstrate that viral IL-10 is a key regulator of successful host immune responses to RhCMV. Viral IL-10 is, therefore, an important target for vaccine strategies against cytomegalovirus (CMV). Furthermore, given the immunoregulatory function of viral IL-10, targeting this protein may prove synergistic with other vaccine therapies and targets. Our study also provides additional evidence that the earliest host-CMV interactions can have a significant impact on the nature of persistent infection.

Engineered Nanoparticle Applications for Recombinant Influenza Vaccines.

Influenza viruses cause seasonal epidemics and represent a pandemic risk. With current vaccine methods struggling to protect populations against emerging strains, there is a demand for a next-generation flu vaccine capable of providing broad protection. Recombinant biotechnology, combined with nanomedicine techniques, could address this demand by increasing immunogenicity and directing immune responses toward conserved antigenic targets on the virus. Various nanoparticle candidates have been tested for use in vaccines, including virus-like particles, protein and carbohydrate nanoconstructs, antigen-carrying lipid particles, and synthetic and inorganic particles modified for antigen presentation. These methods have yielded some promising results, including protection in animal models against antigenically distinct influenza strains, production of antibodies with broad reactivity, and activation of potent T cell responses. Based on the evidence of current research, it is feasible that the next generation of influenza vaccines will combine recombinant antigens with nanoparticle carriers.

Novel strategy for expression and characterization of rabies virus glycoprotein.

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few µg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection.

Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus.

In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.

A Nanostructured Lipid Carrier for Delivery of a Replicating Viral RNA Provides Single, Low-Dose Protection against Zika.

Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago,1 the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials.2 Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront,3 but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines.4 Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness.5 Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.

Oral immunization with a Lactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COE antigen of PEDV.

AIMS: The aims of this study were to develop an effective M cell-targeting oral vaccine, involving Lactobacillus casei to deliver the porcine epidemic diarrhoea virus (PEDV) core neutralizing epitope (COE) antigen conjugated with M cell-targeting peptide Co1 as an adjuvant, against PEDV infection. METHODS AND RESULTS: Genetically engineered L. casei 393 (L393) strains expressing PEDV COE antigen only (pPG-COE/L393) or fused-expressing COE and M cell-targeting peptide Co1 (pPG-COE-Co1/L393) were constructed, and the immunogenicity upon administration as an oral vaccine was evaluated. The results showed that higher anti-PEDV serum IgG and mucosal SIgA antibody responses were induced in mice orally immunized with strain pPG-COE-Co1/L393 as compared to the mice immunized with strain L393 expressing COE alone or carrying the empty plasmid. In addition, the use of the Co1 ligand elicited a splenocyte proliferative response more effectively in comparison with the COE antigen alone and supported a skewed T helper 2 type of immune response against PEDV. CONCLUSIONS: pPG-COE-Co1/L393 can effectively induce mucosal, humoural and Th2-type cellular immune responses against PEDV infection via oral administration. Furthermore, M cell-targeting peptide ligand Co1 is a good mucosal adjuvant. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus casei delivering the COE antigen of PEDV conjugated with a M cell-targeting peptide Co1 as an immune adjuvant is a promising oral vaccine candidate for PEDV.

CD11c-specific bio-nanocapsule enhances vaccine immunogenicity by targeting immune cells.

BACKGROUND: Various nanocarriers have been used to deliver subunit vaccines specifically to dendritic cells (DCs) for the improvement of immunogenicity. However, due to their insufficient DC priming ability, these vaccines could not elicit effective innate immunity. We have recently developed a DC-targeting bio-nanocapsule (BNC) by displaying anti-CD11c IgGs via protein A-derived IgG Fc-binding Z domain on the hepatitis B virus envelope L protein particles (α-DC-ZZ-BNC). RESULTS: After the chemical modification with antigens (Ags), the α-DC-ZZ-BNC-Ag complex could deliver Ags to DCs efficiently, leading to effective DC maturation and efficient endosomal escape of Ags, followed by Ag-specific T cell responses and IgG productions. Moreover, the α-DC-ZZ-BNC modified with Japanese encephalitis virus (JEV) envelope-derived D3 Ags could confer protection against 50-fold lethal dose of JEV injection on mice. CONCLUSION: The α-DC-ZZ-BNC-Ag platform was shown to induce humoral and cellular immunities effectively without any adjuvant.

Adjuvant activity of ethanol extract of Hippophae rhamnoides leaves with inactivated rabies virus antigen.

CONTEXT: Hippophae rhamnoides L. (Elaeagnaceae), commonly known as seabuckthorn (SBT), is known for its medicinal and nutritional properties. OBJECTIVE: Evaluation of in vivo adjuvant activity of SBT leaf extract (SBTE) with inactivated rabies virus antigen (Rb). MATERIALS AND METHODS: Swiss albino mice were immunized with aqueous-alcoholic SBTE (100 mg/kg body weight) or algel (aluminium hydroxide gel) with or without Rb (5% v/v). After priming, booster was administered on day 14. Rabies virus neutralizing antibody (RVNA) titers were estimated by rapid fluorescent focus inhibition test in sera samples collected on days 7, 14, 21, 28 and 35. Effect of adjuvant administration on cytotoxic T lymphocytes (CTLs), memory T cells, plasma and CD11c+ cells was studied by flow cytometry. In vitro hemolysis was assayed in human RBC. RESULTS: RVNA titers were significantly enhanced (p < 0.05) after booster administration in mice immunized with SBTE + Rb as compared to the controls. In combination, SBTE, algel and Rb, enhanced the RVNA titers. CTLs significantly increased (p < 0.05) in SBTE + Rb immunized mice. Memory T cells and plasma cells were 27.9 and 15.9%, respectively, in SBTE + Rb immunized mice as compared to that of 20.3 and 11.3%, respectively, in Rb immunized group. SBTE + Rb enhanced peritoneal CD11c+ cells (25.8%) as compared to 9.4% cells in Rb immunized mice, showed 3.2-fold increment in LPS induced IL-1ß. No RBC hemolysis was observed with SBTE. CONCLUSIONS: This study demonstrates the potential adjuvant activity of SBTE with Rb by increasing RVNA titers and CTL response.

T regulatory cells are critical for the maintenance, anamnestic expansion and protection elicited by vaccine-induced CD8 T cells.

T regulatory (Treg) cells are critical for preventing autoimmunity and suppressing immune responses during cancer and chronic infection. However, the role of Treg cells in the generation of vaccine-induced immune memory remains ill-defined. Using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that transient absence of Treg cells during effector to memory CD8 T-cell transition results in a permanent impairment in the maintenance, function and recall capacity of CD8 T cells. Memory CD8 T cells in mice that were transiently depleted of Treg cells exhibited defective up-regulation of memory markers with a significant decrease in polyfunctionality. However, Treg-depleted mice showed no significant change in CD4 T-cell responses, and antibody levels relative to control. Altogether, this study evaluates the role of Treg cells in the formation of immune memory and demonstrates an important role for Treg cells in promoting memory CD8 T-cell differentiation and vaccine-induced immune protection against intracellular pathogens.

Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs.

A subunit protein vaccine candidate based on norovirus (NoV) virus-like particles (VLPs) and rotavirus (RV) VP6 protein against acute childhood gastroenteritis has been proposed recently. RV VP6 forms different oligomeric nanostructures, including tubes and spheres when expressed in vitro, which are highly immunogenic in different animal models. We have shown recently that recombinant VP6 nanotubes have an adjuvant effect on immunogenicity of NoV VLPs in mice. In this study, we investigated if the adjuvant effect is dependent upon a VP6 dose or different VP6 structural assemblies. In addition, local and systemic adjuvant effects as well as requirements for antigen co-delivery and co-localization were studied. The magnitude and functionality of NoV GII.4-specific antibodies and T cell responses were tested in mice immunized with GII.4 VLPs alone or different combinations of VLPs and VP6. A VP6 dose-dependent adjuvant effect on GII.4-specific antibody responses was observed. The adjuvant effect was found to be strictly dependent upon co-administration of NoV GII.4 VLPs and VP6 at the same anatomic site and at the same time. However, the adjuvant effect was not dependent on the types of oligomers used, as both nanotubes and nanospheres exerted adjuvant effect on GII.4-specific antibody generation and, for the first time, T cell immunity. These findings elucidate the mechanisms of VP6 adjuvant effect in vivo and support its use as an adjuvant in a combination NoV and RV vaccine.

The Influence of Cytomegalovirus on Expression of HLA-G and its Ligand KIR2DL4 by Human Peripheral Blood Leucocyte Subsets.

HLA-G is a non-classical class I HLA antigen, normally expressed in high levels only on extravillous cytotrophoblast. It has immunosuppressive properties in pregnancy and has also been found to be upregulated on leucocytes in viral infection. In this study, proportions of all leucocyte subsets expressing HLA-G were found to be low in healthy subjects positive or negative for cytomegalovirus (CMV). Significantly greater proportions of CD4+ CD69+ and CD56+ T cells expressed HLA-G compared to other T cells. However, following stimulation with CMV antigens or intact CMV, proportions of CD4+, CD8+, CD69+ and CD56+ T cells, and also B cells expressing HLA-G, were significantly increased in CMV+ subjects. Despite some subjects having alleles of HLA-G associated with high levels of expression, no relationship was found between HLA-G genotype and expression levels. Purified B cells from CMV+ subjects stimulated in mixed culture with CMV antigens showed significantly increased HLA-G mRNA expression by real-time polymerase chain reaction. Serum levels of soluble HLA-G were similar in CMV- and CMV+ subjects but levels in culture supernatants were significantly higher in cells from CMV+ than from CMV- subjects stimulated with CMV antigens. The HLA-G ligand KIR2DL4 was mainly expressed on NK cells and CD56+ T cells with no differences between CMV+ and CMV- subjects. Following stimulation with IL-2, an increase in the proportion of CD56+ T cells positive for KIR2DL4 was found, together with a significant decrease in CD56dimCD16+ NK cells. The results show that CMV influences HLA-G expression in healthy subjects and may contribute to viral immune evasion.

The intranasal adjuvant Endocine™ enhances both systemic and mucosal immune responses in aged mice immunized with influenza antigen.

Despite availability of annual influenza vaccines, influenza causes significant morbidity and mortality in the elderly. This is at least in part a result of immunosenescence; the age-dependent decrease in immunological competence that results in greater susceptibility to infections and reduced responses to vaccination. To improve protective immune responses in this age group, new vaccines strategies, such as the use of adjuvants, are needed. Here, we evaluated the mucosal vaccine adjuvant Endocine™, formulated with split influenza antigen and administered intranasally in aged (20-month old) mice. Humoral immune responses were assessed and compared to unadjuvanted intranasal and subcutaneous vaccines. We show that formulation with Endocine™ significantly enhances hemagglutination inhibition (HI) titers, as well as serum IgG and mucosal IgA antibody titers, compared to both types of unadjuvanted vaccines. Thus, our results indicate that intranasal vaccination with Endocine™ is a possible approach for the development of mucosal influenza vaccines for the elderly.

Oral Gavage Delivery of PR8 Antigen with ß-Glucan-Conjugated GRGDS Carrier to Enhance M-Cell Targeting Ability and Induce Immunity.

Oral gavage is known as one of most convenient routes for therapeutic administration in comparison with other available routes such as intravenous, intra muscular, suppository, etc. An oral vaccine delivery system has additional potential as it may provide a convenient way to prevent infectious diseases by introducing optimum immunization in mucus. Although oral vaccine delivery has attracted tremendous interest in vaccine delivery research, various limitations have prevented its rate of progress up to the level that was initially expected. However, the major problems of oral vaccine delivery are vaccine instability and lack of absorbability, resulting from degradation of the sophisticated antigens in the acidic medium in the stomach. In order to obtain adequate microfold-cell (M-cell) targeting and uptake, the therapeutic material is required to pass through the stomach and reach the small intestine without degradation. In this project, we have introduced a conjugate of ß-glucan and Glycine-Arginine-Glycine-Aspartic acid-Serine (GRGDS) that is effective for simultaneous protection of the antigen (PR8) and M-cell targeting. According to the experimental results, the cationic ß-glucan-GRGDS conjugate can encapsulate a certain amount of anionic PR8 through electrostatic interaction, which forms nanoparticles with a range of diameter of 200-250 nm. Also, the PR8 incorporated nanoparticles showed high cell viability and stability in diverse environments. Finally, excellent M-cell targeting ability was verified in an in vitro M-cell model. Most importantly, the in vivo test obviously demonstrated the superiority of this system, which significantly increases antibody concentration in serum, intestine, and mucus as measured 21 days after immunization.

Intracellular delivery of HA1 subunit antigen through attenuated Salmonella Gallinarum act as a bivalent vaccine against fowl typhoid and low pathogenic H5N3 virus.

Introduction of novel inactivated oil-emulsion vaccines against different strains of prevailing and emerging low pathogenic avian influenza (LPAI) viruses is not an economically viable option for poultry. Engineering attenuated Salmonella Gallinarum (S. Gallinarum) vaccine delivering H5 LPAI antigens can be employed as a bivalent vaccine against fowl typhoid and LPAI viruses, while still offering economic viability and sero-surveillance capacity. In this study, we developed a JOL1814 bivalent vaccine candidate against LPAI virus infection and fowl typhoid by engineering the attenuated S. Gallinarum to deliver the globular head (HA1) domain of hemagglutinin protein from H5 LPAI virus through pMMP65 constitutive expression plasmid. The important feature of the developed JOL1814 was the delivery of the HA1 antigen to cytosol of peritoneal macrophages. Immunization of chickens with JOL1814 produced significant level of humoral, mucosal, cellular and IL-2, IL-4, IL-17 and IFN-γ cytokine immune response against H5 HA1 and S. Gallinarum antigens in the immunized chickens. Post-challenge, only the JOL1814 immunized chicken showed significantly faster clearance of H5N3 virus in oropharyngeal and cloacal swabs, and 90% survival rate against lethal challenge with a wild type S. Gallinarum. Furthermore, the JOL1814 immunized were differentiated from the H5N3 LPAI virus infected chickens by matrix (M2) gene-specific real-time PCR. In conclusion, the data from the present showed that the JOL1814 can be an effective bivalent vaccine candidate against H5N3 LPAI and fowl typhoid infection in poultry while still offering sero-surveillance property against H5 avian influenza virus.

Fine-Needle Aspiration Biopsy of the Lymph Node: A Novel Tool for the Monitoring of Immune Responses after Skin Antigen Delivery.

Assessment of immune responses in lymph nodes (LNs) is routine in animals, but rarely done in humans. We have applied minimally invasive ultrasound-guided fine-needle aspiration of the LN to a before-and-after study of the immune response to intradermally delivered Ag in healthy volunteers (n = 25). By comparison with PBMCs from the same individual, LN cells (LNCs) were characterized by reduced numbers of effector memory cells, especially CD8(+) TEMRA cells (3.37 ± 1.93 in LNCs versus 22.53 ± 7.65 in PBMCs; p = 0.01) and a marked increased in CD69 expression (27.67 ± 7.49 versus 3.49 ± 2.62%, LNCs and PBMCs, respectively; p < 0.0001). At baseline, there was a striking absence of IFN-γ ELISPOT responses to recall Ags (purified protein derivative, Tetanus toxoid, or flu/EBV/CMV viral mix) in LN, despite strong responses in the peripheral blood. However, 48 h after tuberculin purified protein derivative administration in the ipsilateral forearm resulting in a positive skin reaction, a clear increase in IFN-γ ELISPOT counts was seen in the draining LN but not in PBMCs. This response was lost by 5 d. These data suggest that the low levels of effector memory cells in the LN may explain the low background of baseline ELISPOT responses in LNs as compared with PBMCs, and the appearance of a response after 48 h is likely to represent migration of effector memory cells from the skin to the LN. Hence, it appears that the combination of intradermal Ag administration and draining LN sampling can be used as a sensitive method to probe the effector memory T cell repertoire in the skin.

Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens.

Avian influenza virus (AIV) is spreading worldwide and is a serious threat to the health of poultry and humans. In many countries, low pathogenic AIVs, such as H9N2, have become an enormous economic burden on the commercial poultry industry because they cause mild respiratory disease and decrease egg production. A recombinant Lactobacillus plantarum NC8 strain expressing NP-M1-DCpep from H9N2 AIV has been studied in a mouse model. However, it remains unknown whether this L. plantarum strain can induce an immune response and provide protection against H9N2 AIV in chickens. In this study, chickens that were orally vaccinated with NC8-pSIP409-NP-M1-DCpep exhibited significantly increased T cell-mediated immune responses and mucosal sIgA and IgG levels, which provided protection against H9N2 AIV challenge. More importantly, compared with oral administration of NC8-pSIP409-NP-M1-DCpep, intranasal administration induced stronger immune responses and provided effective protection against challenge with the H9N2 virus by reducing body weight loss, lung virus titers, and throat pathology. Taken together, these findings suggest that L. plantarum expressing NP-M1-DCpep has potential as a vaccine to combat H9N2 AIV infection.

Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge.

Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order to protect against multiple virus strains. We used our self-assembling protein nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs, we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose-dependent manner. Chickens vaccinated with the self-adjuvanted SAPNs induce significantly higher levels of antibodies than those with unadjuvanted SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted SAPNs, mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted SAPN with a great potential as a universal influenza vaccine.