Antibodies In Primates Research Articles

Development of anti-drug antibodies in infant and maternal non-human primates in post-natal development studies

Development of anti-drug antibodies in infant and maternal non-human primates in post-natal development studies

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.

Gonadal Expression of Foxo1, but Not Foxo3, Is Conserved in Diverse Mammalian Species1

The Foxos are key effectors of the PI3K/Akt signaling pathway and regulate diverse physiologic processes. Two of these factors, Foxo1 and Foxo3, serve specific roles in reproduction in the mouse. Foxo3 is required for suppression of primordial follicle activation in females, while Foxo1 regulates spermatogonial stem cell maintenance in males. In the mouse ovary, Foxo1 is highly expressed in somatic cells (but not in oocytes), suggesting an important functional role for Foxo1 in these cells. Given that invertebrate model species such as Caenorhabditis elegans and Drosophila melanogaster harbor a single ancestral Foxo homolog, these observations suggest that gene duplication conferred a selective advantage by permitting the Foxos to adopt distinct roles in oogenesis and spermatogenesis. Our objective was to determine if the remarkably specific expression patterns of Foxo1 and Foxo3 in mouse gonads (and, by inference, Foxo function) are conserved in diverse mammalian species. Western blotting was used to validate isoform-specific antibodies in rodents, companion animals, farm animals, nonhuman primates, and humans. Following validation of each antibody, immunohistochemistry was performed to ascertain Foxo1 and Foxo3 gonadal expression patterns. While Foxo1 expression in spermatogonia and granulosa cells was conserved in each species evaluated, Foxo3 expression in oocytes was not. Our findings suggest that Foxo3 is not uniquely required for primordial follicle maintenance in nonrodent species and that other Foxos, particularly Foxo1, may contribute to oocyte maintenance in a functionally redundant manner.

Frequency of Toxoplasma gondii antibodies in tufted capuchin monkeys (Cebus apella nigritus) from an ecological station in the State of São Paulo, Brazil

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite protozoan. A large percentage of animals presents specific antibodies caused by a previous exposition, resulting in a chronic infection. Felides are the definitive hosts and the other warm-blooded animals, including primates, are the intermediate hosts. This study was aimed to determine the prevalence of T. gondii infection in free-living tufted capuchin monkeys (Cebus apella nigritus) from an ecological station located on Mata de Santa Teresa, Ribeirão Preto, SP, Brazil. T. gondii antibodies were analyzed by modified agglutination test (MAT) in serum samples of 36 tufted capuchin monkeys, considering eight as cut-off titer. From the studied animals, 3/36 (8.33%; CI95% 3.0-21.9%) presented T. gondii antibodies, all with titer 32. No significative difference was observed relating to the sex (1/3 male and 2/3 female), and to the age (1/3 young and 2/3 adult) (P>0.05). Thus, these results demonstrate the presence of T. gondii antibodies in primates from São Paulo state.

Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

Puumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model.

Development, validation and use of ELISA for antibodies to human alpha-1 antitrypsin

AbstractEvaluation of human antibody responses to alpha-1 antitrypsin (AAT) in clinical trials and clinical practice has been limited by the lack of a validated assay. Here we describe the development and validation of an ELISA method for quantification of human and nonhuman primate antibody responses to human AAT. A reference anti-human AAT serum standard was generated using sera from a cynomolgus macaque injected with a recombinant adeno-associated virus vector expressing human AAT. The ELISA was validated for use with human serum dilutions as low as 1:10 and was able to distinguish between specific responses in cynomolgus serum and non-specific increases in apparent antibody titer in serum from subjects in a clinical trial of an AAT gene therapy vector.

Priming with DNA encoding functional trimers and boosting with soluble trimeric protein elicited tier 2 neutralizing antibodies in non-human primates

Background All known HIV-1 gp120-directed broadly neutralizing antibodies efficiently recognize fully cleaved JR-FL spikes, however, the non-neutralizing gp120-directed antibodies only recognize the uncleaved JR-FL spikes. Therefore, as an immunogen, cleaved, functional spikes may selectively present neutralizing epitopes to B cells, perhaps more efficiently eliciting neutralizing antibodies. However, attempts to make soluble versions of Env that fully mimic the viral spike are yet to be successful. In vitro, this plasmid encodes for fully cleaved, cell-surface, trimeric JR-FL Env spikes, but only when expressed from a mini-LTR in a non-codon optimized state. In vivo, the LTR-driven, non-codon-optimized Env plasmid DNA, likely requires tat co-transfection in trans, and rev expression in cis, for efficient Env expression. Methods To present functional, cleaved spikes to the immune system, we inoculated non-human primates (NHPs) with non-codon optimized JR-FL Env expressing plasmid DNA along with plasmid expressing Tat protein by electroporation. DNA priming was followed by boosts with soluble JR-FL gp140 trimers in adjuvant. As controls codon-optimized and CMV-driven Env plasmids were used to prime and three animals received protein in adjuvant only. Results We report that the DNA priming elicited reasonable ELISA binding titers in NHPs and that the elicitation of neutralizing antibodies against Tier 1 viruses. Following trimer protein boosting of the DNA-primed animals, reasonable titers of Tier 2 neutralizing antibodies in the A3R5 assay were elicited. For the Tier 2 isolates tested to date, much of the neutralizing activity in sera maps to the CD4 binding site. Conclusion

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo.

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

Maximizing tumour exposure to anti‐neuropilin‐1 antibody requires saturation of non‐tumour tissue antigenic sinks in mice

Neuropilin-1 (NRP1) is a VEGF receptor that is widely expressed in normal tissues and is involved in tumour angiogenesis. MNRP1685A is a rodent and primate cross-binding human monoclonal antibody against NRP1 that exhibits inhibition of tumour growth in NPR1-expressing preclinical models. However, widespread NRP1 expression in normal tissues may affect MNRP1685A tumour uptake. The objective of this study was to assess MNRP1685A biodistribution in tumour-bearing mice to understand the relationships between dose, non-tumour tissue uptake and tumour uptake. Non-tumour-bearing mice were given unlabelled MNRP1685A at 10 mg·kg(-1) . Tumour-bearing mice were given (111) In-labelled MNRP1685A along with increasing amounts of unlabelled antibody. Blood and tissues were collected from all animals to determine drug concentration (unlabelled) or radioactivity level (radiolabelled). Some animals were imaged using single photon emission computed tomography - X-ray computed tomography. MNRP1685A displayed faster serum clearance than pertuzumab, indicating that target binding affected MNRP1685A clearance. I.v. administration of (111) In-labelled MNRP1685A to tumour-bearing mice yielded minimal radioactivity in the plasma and tumour, but high levels in the lungs and liver. Co-administration of unlabelled MNRP1685A with the radiolabelled antibody was able to competitively block lungs and liver radioactivity uptake in a dose-dependent manner while augmenting plasma and tumour radioactivity levels. These results indicate that saturation of non-tumour tissue uptake is required in order to achieve tumour uptake and acceptable exposure to antibody. Utilization of a rodent and primate cross-binding antibody allows for translation of these results to clinical settings.

Innate cellular immunity and xenotransplantation.

This review assesses the recent progress in xenograft rejection by innate immune responses, with a focus on innate cellular xenoreactivity. Current literature was reviewed for new insights into the role of innate cellular immunity in xenograft rejection. Increasing evidence confirms that vigorous innate immune cell activation is accounted for by a combination of xenoantigen recognition by activating receptors, and incompatibility in inhibitory receptor-ligand interactions. Although both innate humoral and cellular xenoimmune responses are predominantly elicited by preformed and induced xenoreactive antibodies in nonhuman primates following porcine xenotransplantation, innate immune cells can also be activated by xenografts in the absence of antibodies. The latter antibody-independent response will likely persist in recipients even when adaptive xenoimmune responses are suppressed. In addition to xenograft rejection by recipient innate immune cells, phagocytic cells within liver xenografts are also deleterious to recipients by causing thrombocytopenia. Strategies of overcoming innate immune responses are required for successful clinical xenotransplantation. In addition to developing better immunosuppressive and tolerance induction protocols, endeavors towards further genetic modifications of porcine source animals are ultimately important for successful clinical xenotransplantation.

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.

Antibody Targeting of TGF-β in Cancer Patients

The role of TGF-β in tumor development and progression is complex. Genetic mutations that disrupt the antiproliferative signaling effects of TGF-β play a key role in the process of malignant transformation for many types of tumors. Paradoxically, this loss of sensitivity to TGF-β's inhibitory actions often leads to TGF-β overexpression by the tumor cells or by normal cells that are recruited to the tumor microenvironment. Elevated concentrations of TGF-β in the tumor microenvironment have been shown to facilitate tumor growth and metastasis. Numerous published studies have provided evidence that inhibition of TGF-β using antibodies, soluble receptors and small molecule inhibitors of TGF-β signal transduction can have beneficial effects in murine models of cancer. Given the pleiotropic nature of TGF-β and its homeostatic role in numerous biological processes, serious concerns have been expressed regarding the safety of administering TGF-β antagonists to human patients. Interestingly, the results of numerous animal toxicology studies of TGF-β antibodies in normal rodents and primates have shown that administration of neutralizing anti-TGF-β antibodies is well tolerated and any adverse effects were reversible or self-limiting. Likewise, administration of a human anti-TGF-β antibody (fresolimumab) in three separate human phase 1 clinical trials has also been shown to be well tolerated.

Detection of anti‐Leptospiraantibodies in captive nonhuman primates from Salvador, Brazil

Leptospirosis is a widely distributed zoonosis that affects several species of domestic and wild animals. Under captive conditions, Leptospirosis is a potential problem because the physical conditions in most zoos and research centers cannot prevent the captive animals from being exposed to rodents, raccoons, opossums, and other local wildlife that are known carriers. Yet, despite the potential risk, animals that are destined for reintroduction into the wild are not routinely tested for anti-Leptospira antibodies before their release. The purpose of this study was to determine the occurrence of anti-Leptospira antibodies in captive New World monkeys that were housed in the Wild Animals Screening Center in Salvador, Brazil. Blood samples were collected from 44 monkeys (28 Callithrix jacchus, eight Callithrix pennicilata, and eight Cebus sp.). The animals were screened for antibodies with the microscopic agglutination test. Twenty-five (56.8%) primates were seroreactive, with Icterohaemorrhagiae being the most frequent serogroup. None of the monkeys, however, presented clinical signs of leptospirosis. Thus, seroreactivity with low titers in asymptomatic animals, as observed in this study, suggests exposure to the agent. The unexpected predominance of the serogroup Icterohaemorrhagiae further suggests that exposure to this serogroup occurred in captivity. Therefore, the dangerous possibility cannot be ignored that reintroduced monkeys will carry the leptospiral serovars into wild populations. In conclusion, primates exposed to urban serovars before their release from captivity represent a potentially significant health risk to wild populations.

A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.

Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.

Obtention and Engineering of Non-Human Primate (NHP) Antibodies for Therapeutics

Recombinant antibodies are therapeutic molecules of choice regarding their efficacy, pharmacokinetics and tolerance - all the more if they are human. The efficacy of antibodies generally depends on their affinity for their antigens. The most straightforward approach to isolate human antibodies with high affinities is the construction and screening of human immune libraries, but Humans cannot be immunized with all antigens of interest, for ethical and practical reasons. We circumvented that difficulty by utilizing non-human primates (NHP) instead of Humans and, in the course of our different studies, we have obtained several antibody fragments with high or very high affinities (from 3 nM to 50 pM) and neutralizing properties. The framework regions (FR) - the regions most implicated in tolerance - of these NHP antibody fragments were shown to be very similar to FR encoded by human germline genes. In one case, in a process called "germline humanization" (or "super-humanization"), we have increased that level of similarity and managed to go even beyond the level observed for human antibodies, without any loss of affinity. Here, the methodological peculiarities of our approach, in comparison with immune libraries built from immunised Humans, and the rationales behind these peculiarities, will be reviewed. The isolation of NHP antibody fragments from immune libraries, followed by the "super-humanization" process, opens a new and highly efficient approach for the production of high-quality recombinant antibodies for therapeutic uses.

Newcastle Disease Virus-Vectored Vaccines Expressing the Hemagglutinin or Neuraminidase Protein of H5N1 Highly Pathogenic Avian Influenza Virus Protect against Virus Challenge in Monkeys

H5N1 highly pathogenic avian influenza virus (HPAIV) causes periodic outbreaks in humans, resulting in severe infections with a high (60%) incidence of mortality. The circulating strains have low human-to-human transmissibility; however, widespread concerns exist that enhanced transmission due to mutations could lead to a global pandemic. We previously engineered Newcastle disease virus (NDV), an avian paramyxovirus, as a vector to express the HPAIV hemagglutinin (HA) protein, and we showed that this vaccine (NDV/HA) induced a high level of HPAIV-specific mucosal and serum antibodies in primates when administered through the respiratory tract. Here we developed additional NDV-vectored vaccines expressing either HPAIV HA in which the polybasic cleavage site was replaced with that from a low-pathogenicity strain of influenza virus [HA(RV)], in order to address concerns of enhanced vector replication or genetic exchange, or HPAIV neuraminidase (NA). The three vaccine viruses [NDV/HA, NDV/HA(RV), and NDV/NA] were administered separately to groups of African green monkeys by the intranasal/intratracheal route. An additional group of animals received NDV/HA by aerosol administration. Each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. All groups developed high levels of neutralizing antibodies against homologous and heterologous strains of HPAIV and were protected against challenge with 2 x 10(7) PFU of homologous HPAIV. Thus, needle-free, highly attenuated NDV-vectored vaccines expressing either HPAIV HA, HA(RV), or NA have been developed and demonstrated to be individually immunogenic and protective in a primate model of HPAIV infection. The finding that HA(RV) was protective indicates that it would be preferred for inclusion in a vaccine. The study also identified NA as an independent protective HPAIV antigen in primates. Furthermore, we demonstrated the feasibility of aerosol delivery of NDV-vectored vaccines.

A Potent Malaria Transmission Blocking Vaccine Based on Codon Harmonized Full Length Pfs48/45 Expressed in Escherichia coli

Malaria caused by Plasmodium falciparum is responsible for nearly 1 million deaths annually. Although much progress has been made in the recent past, the development of a safe, effective and affordable malaria vaccine has remained a challenge. A vaccine targeting sexual stages of the parasite will not only reduce malaria transmission by female Anopheles mosquitoes, but also reduce the spread of parasites able to evade immunity elicited by vaccines targeting pre-erythrocytic and erythrocytic asexual stages. We focused our studies on Pfs48/45, a protein expressed in the sexual stages developing within an infected person and one of the most promising transmission-blocking vaccine targets. Functional immunogenicity of Pfs48/45 protein requires proper disulfide bond formation, consequently evaluation of the immunogenicity of recombinant full-length Pfs48/45 has been hampered by difficulties in expressing properly folded protein to date. Here we present a strategy involving harmonization of codons for successful recombinant expression of full length Pfs48/45 in Escherichia coli. The purified protein, designated CH-rPfs48/45, was recognized by monoclonal antibodies directed against reduction-sensitive conformational epitopes in the native protein. Immunogenicity evaluation in mice revealed potent transmission blocking activity in membrane feeding assays of antisera elicited by CH-rPfs48/45 formulated in three different adjuvants, i.e. Alum, Montanide ISA-51 and complete Freund's adjuvant. More importantly, CH-rPfs48/45 formulated with Montanide ISA-51 when administered to nonhuman primates (Olive baboons, Papio anubis) resulted in uniformly high antibody responses (ELISA titers >2 million) in all five animals. Sera from these animals displayed greater than 93% blocking activity in membrane feeding assays after a single immunization, reaching nearly complete blocking after a booster dose of the vaccine. The relative ease of expression and induction of potent transmission blocking antibodies in mice and nonhuman primates provide a compelling rationale and basis for development of a CH-rPfs48/45 based malaria transmission blocking vaccine.

Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone®) increases antibody response, cellular immunity, and antigenically drifted protection

Safe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome–DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8+ cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4+ and CD8+ T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone®—sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone® alone. Mice vaccinated with JVRS-100–Fluzone® and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.

Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising ∼70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria.