Human immune responses to traditional and novel rabies vaccines.

Traditional rabies vaccines given preventatively or after exposure to the virus induce cluster of differentiation 4+ (CD4+) T cell responses that promote the induction of long-lived memory B cells and neutralising antibody-secreting plasma cells. The high cost of rabies vaccines, combined with the complexity of immunisation protocols, is partially to blame for their under-use in exposed individuals and prevents the vaccines' widespread use in preventative childhood immunisation programmes in areas where rabies remains common. Novel vaccines or vaccine adjuvants that reduce the cost of rabies vaccination and afford protective immunity, as well as sustained immunological memory, after a single dose are being developed and may very well reduce the human death toll of rabies.

Les vaccins antirabiques classiques administrés préventivement ou suite à une exposition virale déclenchent une réponse cellulaire des lymphocytes T CD4+ induisant l'activation des lymphocytes B mémoire à longue durée de vie et des plasmocytes sécréteurs d'anticorps neutralisants. Le coût élevé des vaccins contre la rage et la complexité des protocoles de vaccination se traduisent par une sous-utilisation chez les individus exposés et font obstacle à l'emploi généralisé de ces vaccins dans les programmes d'immunisation préventive des enfants dans les régions où la rage est endémique. Des vaccins ou adjuvants innovants sont en cours de développement, qui pourraient réduire les coûts de la vaccination antirabique tout en conférant une immunité protectrice et en renforçant la mémoire immunitaire après l'administration d'une dose unique, ce qui contribuerait à réduire considérablement le tribut en vies humaines payé à la rage.

Las vacunas antirrábicas tradicionales, administradas con fines preventivos o tras la exposición al virus, inducen, en linfocitos T que expresan el cúmulo de diferenciación 4 (linfocitos CD4+), una respuesta que promueve la inducción de células B de memoria de larga vida y células plasmáticas secretoras de anticuerpos neutralizantes. El hecho de que las vacunas antirrábicas se utilicen menos de lo debido en personas expuestas se explica en parte por su elevado costo, que, sumado a la complejidad de los protocolos de inmunización, obstaculiza su empleo generalizado para programas preventivos de inmunización infantil en zonas donde la rabia sigue siendo frecuente. Ahora se están obteniendo adyuvantes o vacunas de nuevo cuño que reducen el costo de la vacunación antirrábica, ofrecen inmunidad protectora y confieren una memoria inmunológica duradera tras una sola dosis, lo que muy bien podría aligerar el duro tributo que se cobra la rabia en vidas humanas.

Adenovirus-mediated artificial MicroRNAs targeting matrix or nucleoprotein genes protect mice against lethal influenza virus challenge.

Influenza virus (IV) infection is a major public health problem, causing millions of cases of severe illness and as many as 500 000 deaths each year worldwide. Given the limitations of current prevention or treatment of acute influenza, novel therapies are needed. RNA interference (RNAi) through microRNAs (miRNA) is an emerging technology that can suppress virus replication in vitro and in vivo. Here, we describe a novel strategy for the treatment of infuenza based on RNAi delivered by a replication-defective adenovirus (Ad) vector, derived from chimpanzee serotype 68 (AdC68). Our results showed that artificial miRNAs (amiRNAs) specifically targeting conserved regions of the IV genome could effectively inhibit virus replication in human embryonic kidney 293 cells. Moreover, our results demonstrated that prophylactic treatment with AdC68 expressing amiRNAs directed against M1, M2 or nucleoprotein genes of IV completely protected mice from homologous A/PR8 virus challenge and partially protected the mice from heterologous influenza A virus strains such as H9N2 and H5N1. Collectively, our data demonstrate that amiRNAs targeting the conserved regions of influenza A virus delivered by Ad vectors should be pursued as a novel strategy for prophylaxis of IV infection in humans and animals.

Adenovirus-based vaccines against rhesus lymphocryptovirus EBNA-1 induce expansion of specific CD8+ and CD4+ T cells in persistently infected rhesus macaques.

UNLABELLED: The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8(+) and CD4(+) T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination. IMPORTANCE: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines.

Donor vigilance data of a blood transfusion service: A multicenter analysis.

BACKGROUND: Donor vigilance is an important part of the quality management system of blood transfusion services. The evaluation of donor side effects helps to improve the donation process and donor compliance. The aim of the present study was to evaluate donor vigilance data in whole blood and plasmapheresis donors of a blood donor service. MATERIALS AND METHODS: Donors fulfilling current national and European eligibility criteria underwent whole blood and plasmapheresis donation (PCS and MCS+ (Haemonetics, Braintree, USA), A 200 (Fenwal, Round Lake, USA). Whole blood was collected at fixed and mobile sites while plasmaphereses were performed at 8 plasma centers. From 2011 to 2013 donor information was provided for gender, age, body weight, height, first and repeat donation. Donors were monitored for venipuncture and circulatory associated side effects. RESULTS: The total incidences of adverse events were 5004 (0.56%) in repeat donors and 2111 (2.78%) in first time donors for whole blood donation and 3323 (1.01%) and 514 (7.96%) for plasmaphereses, respectively. Circulatory associated events were 2679 (0.30%) for whole blood donation and 1624 (0.49%) for plasmaphereses. CONCLUSION: Our donor vigilance data of a blood transfusion service show that whole blood and plasmapheresis are safe with low incidences of adverse events. Repeat donation and age are predictors for low rates of adverse events. On the other hand, first time donation and female gender were associated with higher incidences of adverse events.

CD4+ and CD8+ T-cell responses to latent antigen EBNA-1 and lytic antigen BZLF-1 during persistent lymphocryptovirus infection of rhesus macaques.

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4(+) and/or CD8(+) T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4(+) T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8(+) T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.

Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae.

In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (Ad human serotype 5 [HAdV-5; here referred to as AdHu5]), D (HAdV-26; here referred to as AdHu26), and E (simian serotypes SAdV-23 and SAdV-24; here referred to as chimpanzee serotypes 6 and 7 [AdC6 and AdC7, respectively]) of the Adenoviridae. Seroprevalence rates and titers of neutralizing antibodies to the two human-origin Ads were found to be higher than those reported previously, especially in countries of sub-Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 were markedly lower. Healthy human adults from the United States had readily detectable circulating T cells recognizing Ad viruses, the levels of which in some individuals were unexpectedly high in response to AdHu26. The magnitude of T-cell responses to AdHu5 correlated with those to AdHu26, suggesting T-cell recognition of conserved epitopes. In mice, all of the different Ad vectors induced CD8(+) T-cell responses that were comparable in their magnitudes and cytokine production profiles. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by the AdHu26 and AdC vectors were markedly lower than those induced by the AdHu5 vector. AdHu26 vectors and, to a lesser extent, AdC vectors induced more potent Ad-neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human Ads.

Sendai virus-specific T cell clones. V. Induction of a virus-specific response by antiidiotypic antibodies directed against a T helper cell clone.

We used an antiidiotypic antibody directed against a Sendai virus-specific T helper cell clone to stimulate an immune response in vivo. In addition, the antiidiotypic antibody induced T cells that mediated a delayed-type hypersensitivity (DTH) response in several different mouse strains. Induction of the DTH response by the antiidiotypic antibody, in contrast to the DTH responses induced by virus, demonstrated a remarkable lack of H-2 restriction.

The extracellular matrix of Volvox carteri: molecular structure of the cellular compartment.

The extracellular matrix (ECM) of Volvox contains insoluble fibrous layers that surround individual cells at a distance to form contiguous cellular compartments. Using immunological techniques, we identified a sulfated surface glycoprotein (SSG 185) as the monomeric precursor of this substructure within the ECM. The primary structure of the SSG 185 poly-peptide chain has been derived from cDNA and genomic DNA. A central domain of the protein, 80 amino acid residues long, consists almost exclusively of hydroxyproline residues. The chemical structure of the highly sulfated polysaccharide covalently attached to SSG 185 has been determined by permethylation analysis. As revealed by EM, SSG 185 is a rod-shaped molecule with a 21-nm-long polysaccharide strand protruding from its central region. The chemical nature of the cross-links between SSG 185 monomers is discussed.

Use of mouse anti-rabies monoclonal antibodies in postexposure treatment of rabies.

Immunization of mice and hamsters with a cocktail of mouse MAbs specific for rabies virus nucleocapsid protein and glycoprotein protected animals not only when challenged with a lethal dose of rabies virus after immunization, but also in post-exposure situations. Hamsters treated with the MAb cocktail 3 h after virus inoculation were completely protected from lethal rabies virus infection, and 80% of the animals survived when the MAb cocktail was given 36 h after virus challenge. The potential usefulness of this MAb cocktail for the postexposure treatment of human rabies is discussed.

Comparison of the effects of amino acid substitutions and beta-N- vs. alpha-O-glycosylation on the T-cell stimulatory activity and conformation of an epitope on the rabies virus glycoprotein.

The first potential N-glycosylation site of the rabies virus glycoprotein, the antigen that carries epitopes for glycoprotein-specific T-cells and virus neutralizing antibodies, is glycosylated inefficiently. Recently, we showed that addition of a beta-N-acetyl-glucosamine moiety to the asparagine residue in the corresponding synthetic fragment V V E D E G C T N L S G F (amino acids 29-41), significantly diminished the T-cell stimulatory activity and reduced the characteristic alpha-helicity of the peptide. The amino acid sequence of the glycoprotein in this region exhibits some degree of variability among different rabies virus and rabies virus related strains, including the replacement of the asparagine residue with aspartic acid or threonine. In the current study, stimulation of a specific T-cell clone by various viral strains and appropriate tridecapeptide sequences and their analogs was investigated. The T-cell recognition pattern of the rabies and rabies-related viruses was identical to that of the synthetic peptides representing the respective epitope sequences. While the asparagine could be replaced without complete loss of T-cell stimulatory activity, amino acid modifications at the C-terminus of the peptide were not tolerated. In contrast to glycosylation of the asparagine, coupling of an N-acetyl-galactosamine moiety at the serine, or galactosyl-N-acetyl-galactosamine moieties at the threonines preceding or replacing the asparagine (all O-linked sugars in the natural alpha-anomeric configuration) resulted in epitopes that lowered rather than abolished the T-cell stimulatory activity. All non-glycosylated peptides assumed a low-to-medium helicity in trifluoroethanol. O-glycosylation was more efficient than N-glycosylation in breaking the helical conformation of the peptides to result in the formation of reverse-turns or unordered structure.

The effects of post-translational side-chain modifications on the stimulatory activity, serum stability and conformation of synthetic peptides carrying T helper cell epitopes.

Peptides 31D and VF13, corresponding to the rabies virus nucleo- and glycoproteins, respectively, vigorously stimulate T helper cells of the appropriate specificity. Earlier we showed how internal and external glycosylation affects the major histocompatibility complex molecule (MHC)-binding ability and conformation of these T-cell epitopes (Otvos et al. (1994) Biochim. Biophys. Acta 1224, 68-76; Otvos et al. (1995) Biochim. Biophys. Acta 1267, 55-64). In the current report, we examined the T-helper cell stimulatory ability after introduction of a new set of post-translational modifications. To obtain general information concerning the effects of amino acid side-chain modifications on other biochemical properties of protein fragments, we studied the serum stability and the conformation of the 31D and VF13 peptides. We found that the extent of the reduction of the T-cell stimulatory activity depends upon the location in the sequence of the host amino acid residue. Generally, beta-linked sugars in mid-chain positions had a greater inhibitory effect than alpha-linked sugars attached to identical amino acids. In a case where mid-chain glycosylation just marginally reduced the T-cell stimulatory activity, the beta-linked glycopeptide was significantly more resistant to serum proteases. This finding suggests that addition of beta-linked carbohydrates might be superior to the addition of alpha-linked sugars for vaccine development, and generally for peptide agonist drug design. In addition, data presented here provide the first documentation that phosphorylation and sulfation of tyrosine residues may retain the MHC-binding ability and T-cell stimulatory activity of class II epitopes. The sulfated and the phosphorylated 31D peptides exhibited considerably increased serum stability compared to the unmodified parent peptide. Finally, all post-translational modifications destabilized the dominant alpha-helical or turn structures of the peptides presented in aqueous trifluoroethanol mixtures. While the circular dichroism spectra of the alpha- and beta-linked VF13 glycopeptides with monosaccharides were almost indistinguishable, the structure of the glycopeptides depended upon the length of the sugar moiety. Significantly, incorporation of sulfate or phosphate groups resulted in identical peptide conformations.

Glycosylation of synthetic T helper cell epitopic peptides influences their antigenic potency and conformation in a sugar location-specific manner.

The immunodominant T helper cell epitopes 31D and VF13N of rabies virus nucleoprotein and glycoprotein, respectively, correspond to peptide sequences AVYTRIMMNGGRLKR and VVEDEGCTNLSGF, and are expressed between amino acids 404-418 and 29-41, of the appropriate proteins. We investigated how internal or external glycosylation affects the biological activity and conformation of the peptides 31D and VF13N. Mid-chain incorporation of maltobiose or N-acetylglucosamine moieties into the asparagine residues greatly diminished the T-cell stimulatory activity in vitro (due to the diminished ability of the glycopeptides to bind to major histocompatibility complex determinants) and reduced the characteristic alpha-helicity of the peptides in aqueous trifluoroethanol solutions. In contrast, addition of maltobiose- or N-acetylglucosamine-coupled asparagines to the N-termini of peptides 31D and VF13N resulted in unchanged T-cell activity. Furthermore, N-terminal glycosylation of peptide 31D, as indicated by the functional assay, decreased the sensitivity of the peptide to degradation in human serum and did not affect the alpha-helical conformation. These data indicate that glycosylation of T-cell epitopes is not a preferable method for the preparation of antagonists, but incorporation of the sugars to appropriate positions may be advantageous in the design of T-cell agonists and peptide-based vaccines.

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

Adenoviral gene delivery for HIV-1 vaccination.

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.

New developments in the pre- and post-exposure treatment of rabies.

Two approaches have been made to develop the alleged "second generation rabies vaccines". One utilizes recombinant DNA, expressing the G protein gene in a heterotypic host, the vaccinia virus, and has proven efficacious in protecting animals from rabies infection. The second approach posits that only a portion of the native viral antigen may be required to induce protective response. To define regions of the subunit molecule recognized by different immune effectors, epitopes on the G protein and nucleoprotein were identified which are recognized by B and T cells. These epitopes were delineated using chemically or enzymatically cleaved fragments of the native antigens or overlapping synthetic peptides covering the entire antigenic amino acid sequence. Protein fragments or synthetic peptides evincing antigenic activity were tested for ability to induce virus-specific immune responses in vivo. Immunization with synthetic peptides carrying epitopes for B and T cells conferred solid protection against lethal rabies virus infection.

The use of antiidiotypic antibodies as vaccines against infectious agents.

The development of the antibodies to the combining site of an antigen-binding antibody has been documented in humans as well as experimental animals. These antibodies have been shown in experimental models to have an important regulatory role in their ability to affect both B- and T-cell responses to antigen. Recently, with the development of technology for cloning T cells, it has been possible to produce monoclonal antibodies to the T-cell receptor proteins. These T-cell antiidiotypic antibodies have been shown to activate both B- and T-cell responses. For this reason, such reagents have the potential to be used as nonantigen-containing immunogens. Our work has demonstrated that such monoclonal anti-T-cell antiidiotypes are capable of stimulating immunity to lethal viral infections. The implications of the use of antiidiotypes against infectious organisms are discussed.

DNA vaccines.

Within the last decade bacterial plasmids encoding foreign antigens have revolutionized vaccine design. Although no DNA vaccine has yet been approved for routine human or veterinary use, the potential of this vaccine modality has been demonstrated in experimental animal models. Plasmid DNA vaccination has shown efficacy against viral, bacterial and parasitic infections, modulated the effects of autoimmune and allergic diseases and induced control over cancer progression. With a better understanding of the basic immune mechanisms that govern induction of protective or curative immune responses, plasmid DNA vaccines and their mode of delivery are continuously being optimized. Because of the simplicity and versatility of these vaccines, various routes and modes of delivery are possible to engage the desired immune responses. These may be T or B effector cell responses able to eliminate infectious agents or transformed cells. DNA vaccines may also induce an immunoregulatory/modulatory or immunosuppressive (tolerizing) response that interferes with the differentiation, expansion or effector functions of B and T cells. In this sense a DNA vaccine may be thought of as a 'negative' vaccine. Pre-clinical and initial small-scale clinical trials have shown DNA vaccines in either of these modes to be safe and well tolerated. Although DNA vaccines induce significant immune responses in small animal trials their efficacy in humans has so far been less promising thus necessitating additional optimizations of this novel vaccine approach.

Novel chimpanzee adenovirus-vectored respiratory mucosal tuberculosis vaccine: overcoming local anti-human adenovirus immunity for potent TB protection.

Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette-Guérin (BCG) vaccine. Developing safe and effective respiratory mucosal TB vaccines represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of vaccination. We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cell responses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal immunization, endorsing its further clinical development in humans.

The influence of DNA sequence on the immunostimulatory properties of plasmid DNA vectors.

To determine the influence of DNA sequence on immunostimulatory properties of vaccine vectors, we tested the induction of in vitro and in vivo immune responses by plasmids modified to contain extended runs of dG sequences. Studies with oligonucleotides indicate that dG sequences can directly stimulate B cells as well as enhance the activity of immunostimulatory CpG motifs because of interaction with the macrophage scavenger receptor (MSR); this receptor can bind a variety of polyanions including dG sequences. To modify vectors, we introduced stretches of 20-60 dG residues into the pCMV-beta and pSG5rab.gp vectors and measured the ability of these plasmids to induce IL-12 and IFN-gamma production by murine splenocytes. The induction of in vivo antibody responses to rabies glycoprotein was also assessed with the pSG5rab.gp vectors. In in vitro cultures, cytokine production induced by plasmids with and without dG sequences was similar. Furthermore, the addition of dG sequences to pSG5rab.gp vectors failed to enhance the anti-rabies glycoprotein response to immunization. To assess further mechanisms by which plasmids stimulate macrophages, we measured the effects of MSR ligands on in vitro cytokine induction. In in vitro cultures, poly(G), dG30, and fucoidan inhibited IL-12 induction by plasmids. IL-12 induction was also inhibited by mammalian DNA but was unaffected by polyanions that are not MSR ligands. Together, these results suggest that the addition of 20 to 60-base dG sequences to plasmids does not significantly affect their properties as immunostimulators or vaccines. Furthermore, these results suggest that MSR ligands can block cytokine induction by plasmid DNA whether or not the plasmid contains extended runs of dG.

Insect peptides with improved protease-resistance protect mice against bacterial infection.

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia coli infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.