Protective Efficacy In Mice Research Articles

Comparison of Immunogenicity and Protective Efficacy of the Intranasal and Intraperitoneal Immunization Routes of <i>Escherichia albertii</i> Strain DM104 in Mouse Model

In recent years, our group isolated the Escherichia albertii strain DM104 and characterized it as a vaccine strain against Shigella dysenteriae type 4 in the guinea pig eye model. Protective efficacy of different routes of immunization such as intranasal, oral, and intrarectal routes were also determined and compared by challenging immunized guinea pigs against live S. dysenteriae. In the current study, we compared the intranasal and intraperitoneal routes of immunizations with the DM104 vaccine strain in mice to understand the better route of administration of the DM104 vaccine and its immunogenicity as well as protective efficacy in mouse model. The results indicate that the immune response elicited by the DM104 strain is strongly dependent on the immunization route, with the intranasal route being more effective than the intraperitoneal route following intraperitoneal live S. dysenteriae challenge. Intranasal immunization yielded 80% protective efficacy in immunized mice whereas, intraperitoneal immunization could not provide any protection. Protection generated by intranasal immunization was accompanied by high titre of anti-whole cell lysate IgG and IgA in DM104 immunized sera compared to sera collected from mice of control group. All these data demonstrate the intranasal route of the vaccine DM104 strain in mouse model to be a better immunization route to protect the animals against live S. dysenteriae challenge.
 Bangladesh J Microbiol, Volume 37 Number 2 December 2020, pp 38-41

A Systemic Prime-Intrarectal Pull Strategy Raises Rectum-Resident CD8+ T Cells for Effective Protection in a Murine Model of LM-OVA Infection.

As the entry sites of many pathogens such as human immunodeficiency virus (HIV), mucosal sites are defended by rapidly reacting resident memory T cells (TRM). TRMs represent a special subpopulation of memory T cells that persist long term in non-lymphoid sites without entering the circulation and provide the “sensing and alarming” role in the first-line defense against infection. The rectum and vagina are the two primary mucosal portals for HIV entry. However, compared to vaginal TRM, rectal TRM is poorly understood. Herein, we investigated the optimal vaccination strategy to induce rectal TRM. We identified an intranasal prime–intrarectal boost (pull) strategy that is effective in engaging rectal TRM alongside circulating memory T cells and demonstrated its protective efficacy in mice against infection of Listeria monocytogenes. On the contrary, the same vaccine delivered via either intranasal or intrarectal route failed to raise rectal TRM, setting it apart from vaginal TRM, which can be induced by both intranasal and intrarectal immunizations. Moreover, intramuscular prime was also effective in inducing rectal TRM in combination with intrarectal pull, highlighting the need of a primed systemic T cell response. A comparison of different pull modalities led to the identification that raising rectal TRM is mainly driven by local antigen presence. We further demonstrated the interval between prime and boost steps to be critical for the induction of rectal TRM, revealing circulating recently activated CD8+ T cells as the likely primary pullable precursor of rectal TRM. Altogether, our studies lay a new framework for harnessing rectal TRM in vaccine development.

Will bacille Calmette-Guerin immunization arrest the COVID-19 pandemic?

Will bacille Calmette-Guerin immunization arrest the COVID-19 pandemic?

Efficient Delivery of Dengue Virus Subunit Vaccines to the Skin by Microprojection Arrays.

Dengue virus is the most important arbovirus impacting global human health, with an estimated 390 million infections annually, and over half the world’s population at risk of infection. While significant efforts have been made to develop effective vaccines to mitigate this threat, the task has proven extremely challenging, with new approaches continually being sought. The majority of protective, neutralizing antibodies induced during infection are targeted by the envelope (E) protein, making it an ideal candidate for a subunit vaccine approach. Using truncated, recombinant, secreted E proteins (sE) of all 4 dengue virus serotypes, we have assessed their immunogenicity and protective efficacy in mice, with or without Quil-A as an adjuvant, and delivered via micropatch array (MPA) to the skin in comparison with more traditional routes of immunization. The micropatch contains an ultra-high density array (21,000/cm2) of 110 μm microprojections. Mice received 3 doses of 1 μg (nanopatch, intradermal, subcutaneous, or intra muscular injection) or 10 μg (intradermal, subcutaneous, or intra muscular injection) of tetravalent sE spaced 4 weeks apart. When adjuvanted with Quil-A, tetravalent sE vaccination delivered via MPA resulted in earlier induction of virus-neutralizing IgG antibodies for all four serotypes when compared with all of the other vaccination routes. Using the infectious dengue virus AG129 mouse infectious dengue model, these neutralizing antibodies protected all mice from lethal dengue virus type 2 D220 challenge, with protected animals showing no signs of disease or circulating virus. If these results can be translated to humans, MPA-delivered sE represents a promising approach to dengue virus vaccination.

Vaccination With a Single Consensus Envelope Protein Ectodomain Sequence Administered in a Heterologous Regimen Induces Tetravalent Immune Responses and Protection Against Dengue Viruses in Mice.

The development of a safe and effective tetravalent dengue vaccine that elicits protection against all dengue virus (DENV) serotypes is urgently needed. The consensus sequence of the ectodomain of envelope (E) protein of DENV (cE80) has been examined as an immunogen previously. In the current study, a cE80 DNA (D) vaccine was constructed and evaluated in conjunction with the cE80 protein (P) vaccine to examine whether both vaccines used together can further improve the immune responses. The cE80 DNA vaccine was administrated using either a homologous (DNA alone, DDD) or heterologous (DNA prime-protein boost: DDP or DPP) regimen, and evaluated for immunogenicity and protective efficacy in mice. Among the three DNA-based immunization regimens tested, DDP immunization is the optimal immunization regimen that elicited the greatest systemic immune response and conferred protection against all four DENV serotypes. This work provides innovative ideas for the development of consensus E-based dengue vaccines and the testing of optimal immunization regimens.

Decreases in Both the Seroprevalence of Serum Antibodies and Seroprotection against Japanese Encephalitis Virus among Vaccinated Children.

The incidence of Japanese encephalitis (JE) has significantly decreased in China due to JE vaccines. In this study, we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus (JEV) to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children. A total of 300 serum samples were collected from vaccinated children aged 3-10years in Zhaotong, Yunnan, China. The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test. The highest seropositivity of 82% was observed in vaccinated children during the first 0.5-1.5years after booster vaccination. Then, the seropositivity began to decline and remained lower than the original level observed in the 0.5-1.5-year group. An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination. Similarly, the neutralizing antibody (nAb) titres gradually decreased over time, and the levels showed a positive correlation with the protective efficacy in mice. This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine. Our results provide data that clarify the persistence and waning of antibodies to JEV, which may help elucidate the pathogenesis of JE.

Development of a novel multiepitope chimeric vaccine against anthrax.

Bacillus anthracis (BA), the etiological agent of anthrax, secretes protective antigen (PA), lethal factor (LF), and edema factor (EF) as major virulence mediators. Amongst these, PA-based vaccines are most effective for providing immunity against BA, but their low shelf life limits their usage. Previous studies showed that B-cell epitopes, ID II and ID III present in PA domain IV possess higher toxin neutralization activity and elicit higher antibody titer than ID I. Moreover, N-terminal region of both LF and EF harbors PA-binding sites which share 100% identity with each other. Here, in this study, we have developed an epitope-based chimeric vaccine (ID-LFn) comprising ID II-ID III region of PA and N-terminal region of LF. We have also evaluated its protective efficacy as well as stability and found it to be more stable than PA-based vaccine. Binding reactivities of ID-LFn with anti-PA/LF/EF antibodies were determined by ELISA. The stability of chimeric vaccine was assessed using circular dichroism spectroscopy. ID-LFn response was characterized by toxin neutralization, lymphocyte proliferation isotyping and cytokine profiling. The protective efficacy was analyzed by challenging ID-LFn-immunized mice with B. anthracis (pXO1+ and pXO2+). ID-LFn was found to be significantly stable as compared to PA. Anti-ID-LFn antibodies recognized PA, LF as well as EF. The T-cell response and the protective efficacy of ID-LFn were found to be almost similar to PA. ID-LFn exhibits equal protective efficacy in mice and possesses more stability as compared to PA along with the capability of recognizing PA, LF and EF at the same time. Thus, it can be considered as an improved vaccine against anthrax with better shelf life. ID-LFn, a novel multiepitope chimeric anthrax vaccine: ID-LFn comprises of immunodominant epitopes of domain 4 of PA and N-terminal homologous stretch of LF and EF. The administration of this protein as a vaccine provides protection against anthrax.

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

SummaryIn this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ‐chain fragment and the tailpiece from immunoglobulin μ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB‐PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N‐glycans linked to TB‐PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low‐affinity FcγRs. Immunization studies in mice indicated that TB‐PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen‐IgG γ‐chain molecules with inherent functional characteristics that are desirable in vaccines.

Plant-Produced Subunit Vaccine Candidates against Yellow Fever Induce Virus Neutralizing Antibodies and Confer Protection against Viral Challenge in Animal Models.

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.

CagL from Helicobacter pylori has ADP-ribosylation activity and exerts partial protective efficacy in mice

Mono ADP-ribosyltransferases are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In prokaryotes, mono ADP-ribose transfer enzymes often represent a family of exotoxins that display activity in a variety of bacteria responsible for causing disease in plants and animals. A bioinformatic approach has allowed us to identify that CagL gene from some Helicobacter pylori strains shares a sequence pattern with ADP-ribosylating toxins of the CT-group. In this manuscript we show that recombinant CagL from Shi470 is catalytically active showing ADP-ribosyltransferase, NAD-glycohydrolase, and auto-ADP-ribosylation activities. This is the first time that a catalytically active member of the ADP-ribosyltransferase family is identified in Helicobacter pylori. This observation may lead to the discovery of novel functions exerted by CagL in the pathogenesis of Helicobacter pylori. Indeed, we have shown that vaccination with CagL has protective efficacy in mice indicating that CagL may be considered as potential component of a Helicobacter pylori vaccine.

Evaluation of protective efficacy of three novel H3N2 canine influenza vaccines.

Canine influenza virus (CIV) has the potential risk to spread in different areas and dog types. Thus, there is a growing need to develop an effective vaccine to control CIV disease. Here, we developed three vaccine candidates: 1) a recombinant pVAX1 vector expressing H3N2 CIV hemagglutinin (pVAX1-HA); 2) a live attenuated canine adenovirus type 2 expressing H3N2 CIV hemagglutinin (rCAV2-HA); and 3) an inactivated H3N2 CIV (A/canine/Guangdong/01/2006 (H3N2)). Mice received an initial intramuscular immunization that followed two booster injections at 2 and 4 weeks post-vaccination (wpv). The splenic lymphocytes were collected to assess the immune responses at 6 wpv. The protective efficacy was evaluated by challenging H3N2 CIV after vaccination (at 6 wpv). Our results demonstrated that all three vaccine candidates elicited cytokine and antibody responses in mice. The rCAV2-HA vaccine and the inactivated vaccine generated efficient protective efficacy in mice, whereas limited protection was provided by the pVAX1-HA DNA vaccine. Therefore, both the rCAV2-HA live recombinant virus and the inactivated CIV could be used as potential novel vaccines against H3N2CIV. This study provides guidance for choosing the most appropriate vaccine for the prevention and control of CIV disease.

Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab.

Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site Ø at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site Ø are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site Ø-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen.IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with persistent infection is limited because of cost, determined from the high doses needed to compensate for its relatively low neutralizing potency. Prior efforts to improve the in vitro potency of site II-specific antibodies did not translate to significant in vivo dose sparing. We isolated a pre-F protein-specific, high-potency neutralizing antibody (5C4) that recognizes antigenic site Ø and compared its efficacy to that of the murine precursor of palivizumab (antibody 1129) matched for isotype and pre-F protein binding affinities. Our findings demonstrate that epitope specificity is an important determinant of antibody neutralizing potency, and defining the mechanisms of neutralization has the potential to identify improved products for the prevention and treatment of RSV infection.

Ginseng Stem-and-Leaf Saponin (GSLS)-Enhanced Protective Immune Responses Induced by Toxoplasma gondii Heat Shocked Protein 70 (HSP70) Against Toxoplasmosis in Mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite and is able to infect birds and mammals including humans. In order to find effective antigen-adjuvant combinations that can boost the immunogenicity and protection of antigen vaccines against toxoplasmosis, we examined the protective efficacy in mice immunized with recombinant protein HSP70 when co-administered with ginseng stem-and-leaf saponins (GSLS) isolated from Panax ginseng . All immunized mice produced significantly high levels of specific antibodies against rTgHSP70, and splenocytes from mice presented strong proliferative immune responses. Vaccinated mice displayed a significantly increased percentage of CD4+ and CD8+ T cells, indicating a strong immune response was triggered. The cellular and humoral immune responses were enhanced, which could be reflected of the increased mRNA levels of IFN-γ and IL-4, respectively. Immunization with rTgHSP70 and GSLS prolonged survival time of the treated mice compared to the controls, which died within 6 days after challenge with the virulent T. gondii RH strain. Our data demonstrate that by addition with GSLS, rTgHSP70 induced a strong immune response and provided partial protection against T. gondii ; therefore GSLS could be used as a promising vaccine adjuvant against acute toxoplasmosis.

A subunit vaccine based on rH-NS induces protection against Mycobacterium tuberculosis infection by inducing the Th1 immune response and activating macrophages.

Mycobacterium tuberculosis (Mtb) is a Gram-positive pathogen which causes tuberculosis in both animals and humans. All tested rH-NS formulations induced a specific Th1 response, as indicated by increased production of interferon γ (IFN-γ) and interleukin 2 (IL-2) by lymphocytes in the spleen of mice which were immunized with rH-NS alone or with rH-NS and the adjuvant cyclic GMP-AMP (cGAMP). Serum from mice immunized with rH-NS with or without adjuvant also had higher levels of IL-12p40 and TNF-α, compared with those from control mice immunized with phosphate-buffered saline. Both vaccines increased protective efficacy in mice which were challenged with Mtb H37Rv, as measured by reduced relative CFU counts in the lungs. We found that rH-NS induced the production of TNF-α, IL-6, and IL-12p40, which relied on the activation of mitogen-activated protein kinases by stimulating the rapid phosphorylation of ERK1/2, p38, and JNK, and on the activation of transcription factor NF-κB in macrophages. Additionally, we also found that rH-NS could interact with TLR2 but not TLR4 in pull-down assays. The rH-NS-induced cytokine production from TLR2-silenced RAW264.7 cells was lower than that from BALB/c macrophages. Prolonged exposure (>24 h) of RAW264.7 cells to rH-NS resulted in a significant enhancement in IFN-γ-induced MHC II expression, which was not found in shTLR2-treated RAW264.7 cells. These results suggest that rH-NS is a TLR2 agonist which induces the production of cytokines by macrophages and up-regulates macrophage function.

Coated protein nanoclusters from influenza H7N9 HA are highly immunogenic and induce robust protective immunity

Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.

Coxsackievirus A16-like particles produced in Pichia pastoris elicit high-titer neutralizing antibodies and confer protection against lethal viral challenge in mice

Coxsackievirus A16 (CA16) is a major causative agent of hand, foot and mouse disease (HFMD) which has been affecting millions of young children annually in the Asia–Pacific region over the last seven years. However, no commercial CA16 vaccines are currently available. In the present study, we investigated the expression of virus-like particles (VLPs) of CA16 in Pichia pastoris yeast and their immunogenicity and protective efficacy in mice. We found that CA16-VLPs could be produced at relatively high levels in P. pastoris yeast transformed with a construct co-expressing the P1 and 3CD proteins of CA16. Mice immunized with the yeast-derived CA16-VLPs produced high-titer serum antibodies with potent neutralization effect specifically on CA16. More importantly, passive immunization with the yeast-derived VLPs fully protected neonatal mice against CA16 lethal challenge in both antisera transfer and maternal immunization experiments. Collectively, our results demonstrate that P. pastoris-derived CA16-VLPs represent a promising CA16 vaccine candidate with proven preclinical efficacy and desirable traits for manufacturing at industrial scale.

A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400nm and zeta potential of 40mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41±0.38log10CFU) than traditional vaccine Bacillus Calmette–Guerin (BCG) (6.01±0.33log10CFU) and PBS control (6.53±0.26log10CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein.

Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.

Optimal attenuation of a PR8-derived mouse pathogenic H5N1 recombinant virus for testing antigenicity and protective efficacy in mice

The PR8-based reverse genetics vector system is widely used to generate commercial vaccine strains, but the pathogenicity of PR8-derived recombinant viruses in mice hinders further immunological studies. In the present study, we generated PR8-derived H5N1 recombinant viruses, in which haemagglutinin (HA) and neuraminidase (NA) originated from a mouse-pathogenic H5N1 low pathogenic avian influenza virus (LPAIV), and the non-structural proteins (NS) and polymerase basic protein 2 (PB2) originated from different H9N2 LPAIVs. In contrast to the control H5N1 recombinant virus, harboring six internal genes from PR8, the NS and PB2 recombinant viruses did not cause body weight loss in mice. However, the NS recombinant virus replicated in the lungs of mice. It was more immunogenic than the PB2 recombinant virus to protect efficiently against a lethal challenge of a H5N1 highly pathogenic AIV with 89 and 88% amino acid identity in HA and NA, respectively. Therefore, the NS gene may be useful for generating nonpathogenic and immunogenic PR8-derived recombinant viruses for studies of antigenicity and protective efficacy in mice.

A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice.

The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.