Elicit Antibody Responses Research Articles

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design

SummaryEliciting HIV-1-specific broadly neutralizing antibodies (bNAbs) remains a challenge for vaccine development, and the potential of passively delivered bNAbs for prophylaxis and therapeutics is being explored. We used neutralization data from four large virus panels to comprehensively map viral signatures associated with bNAb sensitivity, including amino acids, hypervariable region characteristics, and clade effects across four different classes of bNAbs. The bNAb signatures defined for the variable loop 2 (V2) epitope region of HIV-1 Env were then employed to inform immunogen design in a proof-of-concept exploration of signature-based epitope targeted (SET) vaccines. V2 bNAb signature-guided mutations were introduced into Env 459C to create a trivalent vaccine, and immunization of guinea pigs with V2-SET vaccines resulted in increased breadth of NAb responses compared with Env 459C alone. These data demonstrate that bNAb signatures can be utilized to engineer HIV-1 Env vaccine immunogens capable of eliciting antibody responses with greater neutralization breadth.

Recombinant flagellins with deletions in domains D1, D2, and D3: Characterization as novel immunoadjuvants

Bacterial flagellin activates the innate immune system and ultimately the adaptive immune system through a Toll-like receptor 5 (TLR5)-dependent signaling mechanism. Given that TLR5 is widely distributed in epithelia, flagellin is currently being developed as a mucosal adjuvant. Flagellin FliC from Salmonella enterica has four domains: the conserved D0 and D1 domains and the hypervariable D2 and D3 domains. The deletion of D3 and partial deletion of D2 in the recombinant FliCΔ174-400 strongly impairs flagellin’s intrinsic antigenicity but does not affect the TLR5-dependent immunostimulation activity, i.e., the capacity to promote innate responses and adaptive responses to co-administered antigens. Here, we describe the development of novel recombinant flagellins with various deletions encompassing all of D2 and D3, and part of D1. Most of the recombinant molecules conserved an α-helical secondary structure that was as resistant to heat denaturation as the native protein. Whereas the recombinant flagellins’ ability to trigger TLR5 varied markedly in vitro, most gave equivalent in vivo TLR5-dependent innate immune responses following intranasal administration of 2 μg of flagellin to mice. Concordantly, the recombinant flagellins were also valuable respiratory adjuvants for eliciting antibody responses to the foreign antigen ovalbumin, although their intrinsic antigenicity was decreased compared to the native flagellin and not increased compared to FliCΔ174-400. Our results show that the additional deletions of D2 and the distal part of D1 of FliCΔ174-400 does not impact on antigenicity and does not significantly modify the immunostimulatory adjuvant activity. Altogether, this study generated a novel set of recombinant flagellin that constitutes a portfolio of TLR5-dependent candidate adjuvants for vaccination.

Coated and Hollow Microneedle-Mediated Intradermal Immunization in Mice with Diphtheria Toxoid Loaded Mesoporous Silica Nanoparticles

PurposeTo examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice.MethodsDT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls.ResultsLB-MSN-DT had an average size of about 670 nm and a zeta potential of −35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles.ConclusionBoth the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.

Differential Antibody Responses to Outer Membrane Proteins Contribute to Differential Immune Protections between Live and Inactivated Vibrio parahemolyticus.

It is widely accepted that live vaccines elicit higher immune protection than inactivated vaccines. However, the mechanisms are largely unknown. Here, an array with 64 recombinant outer membrane proteins of Vibrio parahemolyticus was developed to explore antibody responses of live and inactivated V. parahemolyticus post immunization of the 8th, 12th, 16th and 20th day. Among the 64 outer membrane proteins, 28 elicited antibody generation. They were all detected in live vaccine-induced immunity but only 15 antibodies were found in inactivated vaccine-induced immunity. Passive immunization showed that higher percent survival was detected in live than inactivated vaccine-induced immunities. Active immunization indicated that out of 19 randomly selected outer membrane proteins, 5 stimulated immune protection against V. parahemolyticus infection. Among them, antibodies to VP2309 and VPA0526 were shared in mice immunized by live or inactivated vaccines, whereas antibodies to VPA0548, VPA1745, and VP1667 were only found in mice immunized by live vaccine. In addition, live V. parahemolyticus stimulated earlier antibody response than inactivated bacteria. These results indicate that not all of the outer membrane proteins elicited antibody responses when they work together in the form of live or inactivated bacteria; live vaccine elicits more protective antibodies, which contribute to higher immune protection in live vaccine than inactivated vaccine. Notably, the recombinant proteins might be different from those separated from live bacteria, and they might be different in their immunogenic potencies.

Antibody targeting of a specific region of Pfs47 blocks Plasmodium falciparum malaria transmission

Transmission-blocking vaccines are based on eliciting antibody responses in the vertebrate host that disrupt parasite development in the mosquito vector and prevent malaria transmission. The surface protein Pfs47 is present in Plasmodium falciparum gametocytes and female gametes. The potential of Pfs47 as a vaccine target was evaluated. Soluble full-length recombinant protein, consisting of three domains, was expressed in E. coli as a thioredoxin fusion (T-Pfs47). The protein was immunogenic, and polyclonal and monoclonal antibodies (mAb) were obtained, but they did not confer transmission blocking activity (TBA). All fourteen mAb targeted either domains 1 or 3, but not domain 2 (D2), and immune reactivity to D2 was also very low in polyclonal mouse IgG after T-Pfs47 immunization. Disruption of the predicted disulfide bond in D2, by replacing cysteines for alanines (C230A and C260A), allowed expression of recombinant D2 protein in E. coli. A combination of mAbs targeting D2, and deletion proteins from this domain, allowed us to map a central 52 amino acid (aa) region where antibody binding confers strong TBA (78-99%). This 52 aa antigen is immunogenic and well conserved, with only seven haplotypes world-wide that share 96–98% identity. Neither human complement nor the mosquito complement-like system are required for the observed TBA. A dramatic reduction in ookinete numbers and ookinete-specific transcripts was observed, suggesting that the antibodies are interacting with female gametocytes and preventing fertilization.

Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design

In the past decades protein nanoparticles have successfully been used for vaccine applications. Their particulate nature and dense repetitive subunit organization makes them perfect carriers for antigen surface display and confers high immunogenicity. Nanoparticles have emerged as excellent candidates for vectorization of biological and immunostimulating molecules. Nanoparticles and biomolecular nanostructures such as ferritins or virus like particles have been used as diagnostic and therapeutic delivery systems, in vaccine development, as nanoreactors, etc. Recently, a new class of bacterial protein compartment has been discovered referred to as encapsulin nanocompartment. These compartments have been used for targeted diagnostics, as therapeutic delivery systems and as nanoreactors. Their biological origin makes them conveniently biocompatible and allows genetic functionalization. The aim of our study was to implement encapsulin nanocompartements for simultaneous epitope surface display and heterologous protein loading for rational vaccine design. For this proof-of-concept-study, we produced Thermotoga maritima encapsulin nanoparticles in E. coli. We demonstrated the ability of simultaneous display in our system by inserting Matrix protein 2 ectodomain (M2e) of influenza A virus at the nanoparticle surface and by packaging of a fluorescent reporter protein (GFP) into the internal cavity. Characterization of the nanoparticles by electronic microscopy confirmed homogenously shaped particles of 24 nm diameter in average. The results further show that engineering of the particle surface improved the loading capacity of the heterologous reporter protein suggesting that surface display may induce a critical elastic deformation resulting in improved stiffness. In Balb/c mice, nanoparticle immunization elicited antibody responses against both the surface epitope and the loaded cargo protein. These results confirm the potential of encapsulin nanocompartments for customized vaccine design and antigen delivery.

Antibody responses against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in children with acute respiratory infection with or without nasopharyngeal bacterial carriage

Background: We studied Immunoglobulin G (IgG) antibody responses against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in young children with acute viral type respiratory infection and analyzed the findings in a multivariate model including age, nasopharyngeal carriage of the tested bacteria and pneumococcal vaccination.Methods: We included 227 children aged 6–23 months with acute respiratory infection. Nasopharyngeal aspirates were tested for bacterial carriage through detection of messenger RNA (mRNA) transcript with nCounter analysis. Acute and convalescent serum samples were tested for IgG antibody response against eight pneumococcal proteins, three proteins from H. influenzae and five proteins from M. catarrhalis in a fluorescent multiplex immunoassay.Results: A two-fold or greater increase in antibodies to S. pneumoniae, H. influenzae and M. catarrhalis was detected in 27.8, 9.7 and 14.1%, respectively. Nasopharyngeal carriage of each of the studied bacteria was not associated with antibody response detection against each respective bacterium. Furthermore, neither age nor pneumococcal vaccination were independently associated to detection of antibody response against the studied bacteria. Children who carried H. influenzae had higher frequency of colonization by M. catarrhalis (175 [80.3%] vs. 2 [22.2%]; p < .001) than those without H. influenzae. Also, children with acute otitis media tended to have higher frequency of antibody response to S. pneumoniae.Conclusion: Nasopharyngeal colonization by S. pneumoniae, H. influenzae and M. catarrhalis did not induce significant increases in antibody levels to these bacteria. Carriage of pathogenic bacteria in the nasopharynx is not able to elicit antibody responses to protein antigens similar to those caused by symptomatic infections.

Development of antibody biomarkers of long term and recent dengue virus infections

Dengue virus (DENV) infections elicit antibody responses to the non-structural protein 1 (NS1) that are associated with protection against disease. However, the antibody isotypes and subclasses involved, and their kinetics have not been extensively studied. We characterized the antibody responses to DENV NS1 by enzyme-linked immunosorbent assay (ELISA) in a longitudinal cohort of 266 confirmed dengue cases in Recife, Northeast Brazil. Samples were collected during the febrile phase and up to over 3 years after onset of symptoms. The antibodies investigated [IgA, IgM, total IgG (all subclasses measured together) and each subclass (IgG2, IgG3 and IgG4) measured separately] had distinct kinetic profiles following primary or secondary DENV infections. Of interest, most of these antibodies were consistently detected greater than 6 months after onset of symptoms, except for IgG3. Anti-dengue NS1-specific IgG was consistently detected from the acute phase to beyond 3 years after symptom onset. In contrast, anti-dengue NS1-specific IgG3 was detected within the first week, peaked at week 2–3, and disappeared within 4–6 months after onset of symptoms. The mean duration of the IgG3 positive signal was 149 days (ranging from 126 to 172 days). In conclusion, anti-dengue NS1-specific IgG and IgG3 are potential biomarkers of long-term and recent (less than 6 months) DENV infections, respectively.

Anti-CTLA-4 based therapy elicits humoral immunity to galectin-3 in patients with metastatic melanoma

ABSTRACTThe combination of CTLA-4 blockade ipilimumab (Ipi) with VEGF-A blocking antibody bevacizumab (Bev) has demonstrated favorable clinical outcomes in patients with advanced melanoma. Galectin-3 (Gal-3) plays a prominent role in tumor growth, metastasis, angiogenesis, and immune evasion. Here we report that Ipi plus Bev (Ipi-Bev) therapy increased Gal-3 antibody titers by 50% or more in approximately one third of treated patients. Antibody responses to Gal-3 were associated with higher complete and partial responses and better overall survival. Ipi alone also elicited antibody responses to Gal-3 at a frequency comparable to the Ipi-Bev combination. However, an association of elicited antibody responses to Gal-3 with clinical outcomes was not observed in Ipi alone treated patients. In contrast to being neutralized in Ipi-Bev treated patients, circulating VEGF-A increased by 100% or more in a subset of patients after Ipi treatment, with most having progressive disease. Among the Ipi treated patients with therapy-induced Gal-3 antibody increases, circulating VEGF-A was increased in 3 of 6 nonresponders but in none of 4 responders as a result of treatment. Gal-3 antibody responses occurred significantly less frequently (3.2%) in a cohort of patients receiving PD-1 blockade where high pre-treatment serum Gal-3 was associated with reduced OS and response rates. Our findings suggest that anti-CTLA-4 elicited humoral immune responses to Gal-3 in melanoma patients which may contribute to the antitumor effect in the presence of an anti-VEGF-A combination. Furthermore, pre-treatment circulating Gal-3 may potentially have prognostic and predictive value for immune checkpoint therapy.

Quantitation and Stability of Protein Conjugation on Liposomes for Controlled Density of Surface Epitopes.

The number and spacing of B-cell epitopes on antigens have a profound impact on the activation of B cells and elicitation of antibody responses, the quantitative aspects of which may be utilized for rational design of vaccines. Ni-chelating liposomes have been widely used as protein carriers in experimental studies of vaccine delivery, owing to the convenience and versatility of this conjugation chemistry. However, the epitope number per particle as well as the stability of protein conjugation on liposomes remain far less characterized. Here we have developed quantitative methods to measure the average spatial density of proteins on liposomes using both ensemble and single-molecule techniques and demonstrated their utility using liposomes conjugated with native proteins of two different sizes. These studies revealed that the initial density of protein conjugation on Ni-chelating liposomes can be finely controlled, but the density can decrease over time upon dilution due to the noncovalent nature of Ni-chelation chemistry. These results indicate that an alternative method other than the Ni-chelation chemistry is needed for stable conjugation of epitopes onto liposomes and also suggest a general strategy that can be used to precisely regulate the epitope density on liposomes for B-cell antigen delivery.

Humoral Responses Elicited by Adenovirus Displaying Epitopes Are Induced Independently of the Infection Process and Shaped by the Toll-Like Receptor/MyD88 Pathway.

The use of serotype 5 adenovirus (Ad)-derived vectors in vaccination is confronted to preexisting anti-Ad immunity. Epitope display on Ad capsid is currently being investigated as an alternative approach of vaccination. The present study seeks to better understand virus- and host-related factors controlling the efficacy of this new vaccination approach. In contrast to an Ad vector expressing ovalbumin as a transgene, Ad displaying an ovalbumin-derived B-cell epitope inserted into the fiber protein was able to elicit antibody responses in both Ad-naive and Ad-immune mice. Moreover, introduction of a set of mutations abrogating Ad interaction with its receptors did not modify the virus capacity to elicit a humoral response against the inserted epitope while reducing its capacity to mount antibody responses against the transgene product. Taken as a whole these data indicate that the efficacy of Ad displaying epitopes requires neither Ad binding to its receptors nor the infection process. In addition, the use of genetically deficient mice demonstrated that both toll-like receptor (TLR)/MyD88 and RIG-I/mitochondrial antiviral-signaling (MAVS) innate immunity pathways were dispensable to mount anti-epitope antibody responses. However, they also revealed that TLR/MyD88 pathway but not RIG-I/MAVS pathway controls the nature of antibodies directed against the displayed epitope.

Plasmablast antibody repertoires in elderly influenza vaccine responders exhibit restricted diversity but increased breadth of binding across influenza strains

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70–89) and young (aged 20–29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine.

ABSTRACTAn important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4+ T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4+ T cell responses.IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with recombinant gp120 protein or MVA-expressed gp140 to enhance antibody responses elicited by the GOVX-B11 DNA prime-MVA boost vaccine. We found that both types of immunogen boosts enhanced potentially protective antibody responses, whereas the gp120 protein boosts also increased CD4+ T cell responses. Our data provide important information for HIV vaccine designs that aim for effective and balanced humoral and T cell responses.

Variations in the quality of malaria-specific antibodies with transmission intensity in a seasonal malaria transmission area of Northern Ghana.

IntroductionPlasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission.MethodsThis retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four P. falciparum AMA 1 variants (from the 3D7, FVO, HB3 and CAMP parasite strains) and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared.ResultsThe data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group.ConclusionThe data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains.

Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response.

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.

Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.

Immunization with the Malaria Diversity-Covering Blood-Stage Vaccine Candidate Plasmodium falciparum Apical Membrane Antigen 1 DiCo in Complex with Its Natural Ligand PfRon2 Does Not Improve the In Vitro Efficacy.

The blood-stage malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1) can induce strong parasite growth-inhibitory antibody responses in animals but has not achieved the anticipated efficacy in clinical trials. Possible explanations in humans are the insufficient potency of the elicited antibody responses, as well as the high degree of sequence polymorphisms found in the field. Several strategies have been developed to improve the cross-strain coverage of PfAMA1-based vaccines, whereas innovative concepts to increase the potency of PfAMA1-specific IgG responses have received little attention even though this may be an essential requirement for protective efficacy. A previous study has demonstrated that immunization with a complex of PyAMA1 and PyRON2, a ligand with an essential functional role in erythrocyte invasion, leads to protection from lethal Plasmodium yoelli challenge in an animal model and suggested to extend this strategy toward improved strain coverage by using multiple PfAMA1 alleles in combination with PfRon2L. As an alternative approach along this line, we decided to use PfRon2L in combination with three PfAMA1 diversity covering variants (DiCo) to investigate the potential of this complex to induce more potent parasite growth inhibitory immune response in combination with better cross-strain-specific efficacy. Within the limits of the study design, the ability of the PfAMA1 DiCo-Mix to induce cross-strain-specific antibodies was not affected in all immunization groups, but the DiCo–PfRon2L complexes did not improve the potency of PfAMA1-specific IgG responses.

A Bivalent Anthrax-Plague Vaccine That Can Protect against Two Tier-1 Bioterror Pathogens, Bacillus anthracis and Yersinia pestis.

Bioterrorism remains as one of the biggest challenges to global security and public health. Since the deadly anthrax attacks of 2001 in the United States, Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, gained notoriety and were listed by the CDC as Tier-1 biothreat agents. Currently, there is no Food and Drug Administration-approved vaccine against either of these threats for mass vaccination to protect general public, let alone a bivalent vaccine. Here, we report the development of a single recombinant vaccine, a triple antigen consisting of all three target antigens, F1 and V from Y. pestis and PA from B. anthracis, in a structurally stable context. Properly folded and soluble, the triple antigen retained the functional and immunogenicity properties of all three antigens. Remarkably, two doses of this immunogen adjuvanted with Alhydrogel® elicited robust antibody responses in mice, rats, and rabbits and conferred complete protection against inhalational anthrax and pneumonic plague. No significant antigenic interference was observed. Furthermore, we report, for the first time, complete protection of animals against simultaneous challenge with Y. pestis and the lethal toxin of B. anthracis, demonstrating that a single biodefense vaccine can protect against a bioterror attack with weaponized B. anthracis and/or Y. pestis. This bivalent anthrax–plague vaccine is, therefore, a strong candidate for stockpiling, after demonstration of its safety and immunogenicity in human clinical trials, as part of national preparedness against two of the deadliest bioterror threats.

A Bivalent Heterologous DNA Virus-Like-Particle Prime-Boost Vaccine Elicits Broad Protection against both Group 1 and 2 Influenza A Viruses.

Current seasonal influenza vaccines are efficacious when vaccine strains are matched with circulating strains. However, they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Thus, there is a critical need for developing so-called "universal" vaccines that protect against all influenza viruses. In the present study, we developed a bivalent heterologous DNA virus-like particle prime-boost vaccine strategy. We show that mice immunized with this vaccine were broadly protected against lethal challenge from group 1 (H1, H5, and H9) and group 2 (H3 and H7) viruses, with 94% aggregate survival. To determine the immune correlates of protection, we performed passive immunizations and in vitro assays. We show that this vaccine elicited antibody responses that bound HA from group 1 (H1, H2, H5, H6, H8, H9, H11, and H12) and group 2 (H3, H4, H7, H10, H14, and H15) and neutralized homologous and intrasubtypic H5 and H7 and heterosubtypic H1 viruses and hemagglutinin-specific CD4 and CD8 T cell responses. As a result, passive immunization with immune sera fully protected mice against H5, H7, and H1 challenge, whereas with both immune sera and T cells the mice survived heterosubtypic H3 and H9 challenge. Thus, it appears that (i) neutralizing antibodies alone fully protect against homologous and intrasubtypic H5 and H7 and (ii) neutralizing and binding antibodies are sufficient to protect against heterosubtypic H1, (iii) but against heterosubtypic H3 and H9, binding antibodies and T cells are required for complete survival. We believe that this vaccine regimen could potentially be a candidate for a "universal" influenza vaccine.IMPORTANCE Influenza virus infection is global health problem. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. However, these vaccines do not protect antigenic variants and newly emerging pandemic and outbreak strains. Because of this, there is an urgent need to develop so-called "universal" influenza vaccines that can protect against both current and future influenza strains. In the present study, we developed a bivalent heterologous prime-boost vaccine strategy. We show that a bivalent vaccine regimen elicited broad binding and neutralizing antibody and T cell responses that conferred broad protection against diverse challenge viruses in mice, suggesting that this bivalent prime-boost strategy could practically be a candidate for a "universal" influenza vaccine.