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Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro . ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

Lassa virus (LASV), a member of the Arenaviridae family, causes a viral hemorrhagic fever endemic to West Africa, where as many as 300,000 infections occur per year. Presently, there are no FDA-approved LASV-specific vaccines or antiviral agents, although the antiviral drug ribavirin has shown some efficacy. A recently identified small-molecule inhibitor of arenavirus entry, ST-193, exhibits submicromolar antiviral activity in vitro. To determine the antiviral utility of ST-193 in vivo, we tested the efficacy of this compound in the LASV guinea pig model. Four groups of strain 13 guinea pigs were administered 25 or 80 mg/kg ST-193, 25 mg/kg of ribavirin, or the vehicle by the intraperitoneal (i.p.) route before infection with a lethal dose of LASV, strain Josiah, and continuing once daily for 14 days. Control animals exhibited severe disease, becoming moribund between days 10 and 15 postinfection. ST-193-treated animals exhibited fewer signs of disease and enhanced survival when compared to the ribavirin or vehicle groups. Body temperatures in all groups were elevated by day 9, but returned to normal by day 19 postinfection in the majority of ST-193-treated animals. ST-193 treatment mediated a 2- to 3-log reduction in viremia relative to vehicle-treated controls. The overall survival rate for the ST-193-treated guinea pigs was 62.5% (10/16) compared with 0% in the ribavirin (0/8) and vehicle (0/7) groups. These data suggest that ST-193 may serve as an improved candidate for the treatment of Lassa fever.

The threat of smallpox as a bioweapon and the emerging threat of human monkeypox, among other poxviral diseases, highlight the need for effective poxvirus countermeasures. ST-246, which targets the F13L protein in vaccinia virus and its homologs in other orthopoxvirus species, provides full protection from lethal poxviral disease in numerous animal models and seems to be safe in humans. All previous evaluations of ST-246 efficacy have been in immunocompetent animals. However, the risk of severe poxviral disease is greater in immunodeficient hosts. Here we report on the efficacy of ST-246 in preventing or treating lethal poxviral disease in immunodeficient mice. After lethal challenge with the Western Reserve strain of vaccinia, Nude, SCID, and JHknockout mice additionally depleted of CD4+and CD8+T cells were not fully protected by ST-246, although survival was significantly extended. However, CD4+T cell deficient, CD8+T cell deficient, JHknockout, and JHknockout mice also deficient for CD4+or CD8+T cells survived lethal challenge when treated with ST-246 starting on the day of challenge. Delaying treatment until 72 h after infection reduced ST-246 efficacy in some models but provided full protection from lethal challenge in most. These findings suggest that ST-246 may be effective in controlling smallpox or other pathogenic orthopoxviruses in some immunodeficient human populations for whom the vaccine is contraindicated.

The re-emerging threat of smallpox and the emerging threat of monkeypox highlight the need for effective poxvirus countermeasures. Currently approved smallpox vaccines have unacceptable safety profiles and, consequently, the general populace is no longer vaccinated, leading to an increasingly susceptible population. ST-246, a small-molecule inhibitor of poxvirus dissemination, has been demonstrated in various animal models to be safe and effective in preventing poxviral disease. This suggests that it may also be used to improve the safety of the traditional smallpox vaccine provided that it does not inhibit vaccine-induced protective immunity. In this study, we compared the immune responses elicited by the smallpox vaccine alone or in combination with ST-246 in mice. Normal lesion formation following dermal scarification with the attenuated New York City Board of Health strain (Dryvax), commonly referred to as a vaccine “take”, was not inhibited although severe lesions and systemic disease due to vaccination with the virulent Western Reserve (VV-WR) strain were prevented. The vaccine given with ST-246 did not affect cellular immune responses or neutralizing antibody titers although anti-vaccinia ELISA titers were slightly reduced. Vaccination in combination with ST-246 provided equivalent short- and long-term protection against lethal intranasal challenge with VV-WR when compared to vaccine alone. These results suggest that ST-246 does not compromise protective immunity elicited by the vaccine and provide the basis for future studies examining the efficacy of ST-246 in preventing or treating adverse events due to vaccination.

The HtrA serine protease has been shown to be essential for bacterial virulence and for survival after exposure to many types of environmental and cellular stresses. A Listeria monocytogenes 10403S htrA mutant was found to be sensitive to oxidative and puromycin-induced stress at high temperatures, showed a reduced ability to form biofilms, and was attenuated for virulence in mice.

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junin virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junin, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 μM), selective (concentration of compound that inhibited cell viability by 50% = >40 μM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10× 50% lethal dose (LD 50 ) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10× LD 50 ) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000× LD 50 of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (7 , 5.2 × 10 7 , and 1.8 × 10 5 PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-gamma to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.

The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium Staphylococcus aureus . There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013–5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine if srtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordonii exhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci.

Plasma pharmacokinetics of ST-246, smallpox therapeutic, was evaluated in mice, rabbits, monkeys and dogs following repeat oral administrations by gavage. The dog showed the lowest Tmax of 0.83 h and the monkey, the highest value of 3.25 h. A 2- to 4-fold greater dose-normalized Cmax was observed for the dog compared to the other species. The mouse showed the highest dose-normalized AUC, which was 2-fold greater than that for the rabbit and monkey both of which by approximation, recorded the lowest value. The Cl/F increased across species from 0.05 L/h for mouse to 42.52 L/h for dog. The mouse showed the lowest VD/F of 0.41 L and the monkey, the highest VD/F of 392.95 L. The calculated extraction ratios were 0.104, 0.363, 0.231 and 0.591 for mouse, rabbit, monkey and dog, respectively. The dog showed the lowest terminal half-life of 3.10 h and the monkey, the highest value of 9.94 h. The simple allometric human VD/F and MLP-corrected Cl/F were 2311.51 L and 51.35 L/h, respectively, with calculated human extraction ratio of 0.153 and terminal half-life of 31.20 h. Overall, a species-specific difference was observed for Cl/F with this parameter increasing across species from mouse to dog. The human MLP-corrected Cl/F, terminal half-life, extraction ratios were in close proximity to the observed estimates. In addition, the first-in-humans (FIH) dose of 485 mg, determined from the MLP-corrected allometry Cl/F, was well within the dose range of 400 mg and 600 mg administered in healthy adult human volunteers.

Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro . Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

Although a highly effective vaccine against smallpox, vaccinia virus (VV) is not without adverse events, some of which can be life-threatening, particularly in immunocompromised individuals. We have recently demonstrated that the immunogenicity and protective efficacy of Dryvax(®) in immunocompetent mice is preserved even when co-administered with ST-246, an orally bioavailable small-molecule inhibitor of orthopoxvirus egress and dissemination. In addition, ST-246 markedly reduced the reactogenicity of the smallpox vaccine ACAM2000 and the highly neurovirulent VV strain Western Reserve (VV-WR). Here, we evaluated the impact of ST-246 co-administration on ACAM2000 reactogenicity, immunogenicity, and protective efficacy in seven murine models of varying degrees of humoral and cellular immunodeficiency: BALB/c and B-cell deficient (JH-KO) mice depleted of CD4(+) or CD8(+) or both subsets of T cells. We observed that ST-246 reduced vaccine lesion severity and time to complete resolution in all of the immunodeficient models examined, except in those lacking both CD4(+) and CD8(+) T cells. Although VV-specific humoral responses were moderately reduced by ST-246 treatment, cellular responses were generally comparable or slightly enhanced at both 1 and 6 months post-vaccination. Most importantly, in those models in which vaccination given alone conferred protection against lethal VV challenge, similar levels of protection were observed at both time points when vaccination was given with ST-246. These data suggest that, with the exception of individuals with irreversible, combined CD4(+) and CD8(+) T-cell deficiency, ST-246 co-administered at the time of vaccination may help reduce vaccine reactogenicity--even in those lacking humoral immunity--without impeding the induction of protective immunity.

Mutants of Streptococcus gordonii resistant to 5-fluorodeoxyuridine (FUdR r ) were isolated. Each strain contained a point mutation resulting in the premature termination of the thymidine kinase (TK) open reading frame ( tdk ). In vitro translation of the mutant tdk coding regions resulted in synthesis of truncated TK polypeptides deficient in TK activity.

Smallpox disease is marked by the dissemination of variola virus within the infected host and the appearance of skin lesions or pocks (reviewed in reference 3 and at http://whqlibdoc.who.int/smallpox/9241561106.pdf). The virus is transmitted between humans mainly by aerosol droplets; however, contact with variola virus-contaminated clothing or bedding may contribute to transmission. Once inhaled, variola virus appears to first infect the upper- or lower-respiratory-tract mucosa and spread to and replicate within the local lymph nodes. The virus then disseminates to the spleen and liver via a transient viremia and replicates in reticuloendothelial cells through an average incubation period of 10 to 12 days. During the high-fever prodrome period that follows, a second wave of viremia occurs and results in the dissemination of the virus to mucous membranes of the mouth and pharynx and to the dermal epithelium of the skin. This dissemination leads to the eruption of lesions over the tongue, mouth, and oropharynx and of rashes that start at the face and extremities and may eventually envelope the whole body. Studies performed for 2 to 3 weeks after the onset of fever indicated that infectious virus is excreted in the throat, urine, and conjunctiva of smallpox patients (20). Additionally, the level and duration of secretion was higher in clinically severe (those with hemorrhagic and confluent lesions) cases than less severe (those with discrete lesions) cases (20). Evidence from epidemiological studies further suggests that transmission from smallpox patients to their contacts occurs only from the time of the earliest appearance of rash, and that excretions from the mouth and nose are the most important source of infectious virus for transmission (8). The transmission rate of smallpox to unvaccinated contacts is estimated to be in the range of 37 to 88% (3). Smallpox was declared eradicated from the natural environment in 1980 as a result of a worldwide vaccination campaign conducted by the World Health Organization. Despite the potential threat of the intentional release of variola virus as a biological weapon in the future, routine mass vaccinations are not implemented due to adverse events associated with live vaccinia virus (VV) immunization (6). The occurrence of vaccine-associated adverse events, such as progressive vaccinia, eczema vaccinatum, generalized vaccinia, and postvaccinial encephalitis, suggests that uncontrolled VV dissemination in the vicinity of the vaccination site or to distal sites elsewhere on the body may have serious consequences in vaccinees, especially those that are immunocompromised. In addition, virus shed from the vaccination site and inadvertently transferred to a household contact with a history of atopic dermatitis may lead to a life-threatening case of eczema vaccinatum (26). VV, the prototypic member of the Orthopoxvirus genus, is used to study virus infection, replication, morphogenesis, and dissemination (reviewed in reference 19). After DNA replication in the cytoplasm, two infectious but structurally and functionally different forms of the virus are formed: intracellular mature virus (IMV or MV) and extracellular enveloped virus (EEV or EV). MVs remain confined to the cytoplasm and do not disseminate from the cell until cell lysis occurs. However, MVs that acquire a double membrane from early endosomes or the trans-Golgi network form intracellular enveloped virus (IEV) and get transported to the cell surface. After the fusion of its outer membrane with the plasma membrane, IEV may be either retained as cell-associated enveloped virus (CEV) or released from the cell surface as EV. In vitro, CEV is involved in the cell-to-cell spreading of VV, whereas EV is involved in virus dissemination to nonadjoining nearby or distal cells (2, 15). The 37-kDa protein p37, encoded by the VV F13L gene, plays a critical role in this process, since the deletion of the gene results in the blockade of MV envelopment and the inhibition of plaque formation (2). In vivo, EV is implicated in virus dissemination within the host and has been shown to be released from squamous epithelial cells of the nasal cavity in mice intranasally (i.n.) infected with the IHD-J strain of VV (15, 16). Despite the abundance of MVs in the cytoplasm of nasal squamous epithelial cells, no MVs are detected extracellularly and almost all free extracellular virions are EV, suggesting that the mechanism of virus release in vivo is by the exocytosis of EV, not by cell disruption (16). Although the original form of infectious VV released from infected hosts appears to be EV (not MV), due to the fragility of the EV membrane to withstand environmental factors, it is thought that the more physically stable MV released by the disruption of the EV outer membrane is responsible for the infection of susceptible hosts (22). As such, reducing the amount of EV released from epithelial cells of the respiratory tract might lower the risk of person-to-person transmission of VV by limiting the amount of shed EV available for conversion to MV, thus resulting in the infection of contacts. ST-246 is an orally bioavailable low-molecular-size (376 g/mol) compound that is selectively active against several orthopoxviruses, including VV and ectromelia, cowpox, camelpox, monkeypox, and variola viruses (4, 23, 28). In vitro, ST-246 treatment resulted in a 10-fold reduction of EV formation (without affecting MV production) and in the inhibition of plaque formation and virus-induced cytopathic effect (28). The analysis of ST-246-resistant cowpox virus variants in vitro has revealed that the antiviral activity of ST-246 is targeted against a product of the V061 gene, which is homologous to the VV F13L gene required for MV envelopment (28). In vivo, ST-246 treatment has been shown to protect mice (ectromelia, vaccinia, or cowpox viruses), rabbits (rabbitpox), and ground squirrels (monkeypox) from lethal orthopoxvirus infections (14, 17, 21, 28). ST-246 treatment additionally reduced virus replication in organs such as spleen and liver (17, 28). In nonhuman primates, ST-246 fully protected cynomolgus monkeys from lethal intravenous (i.v.) challenges with monkeypox and variola virus and significantly reduced viral load and lesion formation (9, 10). In this study, we extended on these previous reports and examined the impact of ST-246 treatment on the in vivo dissemination of virus from the site of inoculation to various distal organs in BALB/c mice infected with the Western Reserve strain of VV (VV-WR) by i.n., percutaneous, i.v., subcutaneous (s.c.), and intraperitoneal (i.p.) routes. We also investigated the effect of ST-246 treatment on the level of virus shedding from the nasal and lung cavities of mice challenged i.n. and from vaccine-induced lesions of ACAM2000-vaccinated mice.

The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized withEscherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Several linear B-cell epitopes within the three proteins were recognized by sera from the immunized mice. In addition, protein-specific cellular responses were detected in spleens of immunized mice by a gamma interferon enzyme-linked immunospot assay using peptides derived from each protein. Our data suggest that a subunit vaccine incorporating bacterially expressed IMV- and EEV-specific proteins can be effective in stimulating anti-vaccinia virus immune responses and providing protection against lethal virus challenge.

A novel vaccine (LL-CRR) made from live, nonpathogenicLactococcus lactisthat expresses the conserved C-repeat region (CRR) of M protein fromStreptococcus pyogenesserotype 6 was tested in mice. Nasally vaccinated mice produced CRR-specific salivary immunoglobulin A (IgA) and serum IgG. Subcutaneously vaccinated mice produced CRR-specific serum IgG but not salivary IgA. A combined regimen produced responses similar to the salivary IgA of nasally vaccinated mice and serum IgG of subcutaneously vaccinated mice. Mice vaccinated nasally or with the combined regimen were significantly protected against pharyngeal infection following a nasal challenge withS. pyogenesM serotype 14. Mice vaccinated subcutaneously were not protected against pharyngeal infection. Mice in all three LL-CRR vaccination groups were significantly protected against the lethal effects ofS. pyogenes. Only 1 of 77 challenged mice that were vaccinated with LL-CRR died, whereas 60 of 118 challenged mice that were vaccinated with a control strain or phosphate-buffered saline died. In conclusion, mucosal vaccination with LL-CRR produced CRR-specific salivary IgA and serum IgG, prevented pharyngeal infection withS. pyogenes, and promoted survival.

Several key protein structural attributes were altered in an effort to optimize expression and immunogenicity of a foreign protein (M protein from Streptococcus pyogenes ) exposed on the surface of Streptococcus gordonii commensal bacterial vectors: (i) a shorter N-terminal region, (ii) the addition of a 94-amino-acid spacer, and (iii) the addition of extra C-repeat regions (CRR) from the M6 protein. A decrease in the amount of cell surface M6 was observed upon deletion of 10 or more amino acid residues at the N terminus. On the other hand, reactivity of monoclonal antibody to surface M6 increased with the addition of the spacer adjacent to the proline- and glycine-rich region, and an increase in epitope dosage was obtained by adding another CRR immediately downstream of the original CRR. The results obtained should facilitate the design of improved vaccine candidates using this antigen delivery technology.

The DegP protease, a multifunctional chaperone and protease, has been shown to be essential for virulence in gram-negative pathogens such as Salmonella enterica serovar Typhimurium, Brucella abortus, Yersinia enterocolitica , and Pseudomonas aeruginosa . The function of DegP in pathogenesis appears to be the degradation of damaged proteins that accumulate as a result of the initial host response to infection, which includes the release of reactive oxygen intermediates. Additionally, the DegP protease plays a major role in monitoring and maintaining the Escherichia coli periplasm and influences E. coli pilus biogenesis. We report here the identification of highly homologous enzymes in Streptococcus pyogenes, Streptococcus gordonii, Streptococcus mutans, Staphylococcus aureus , and Enterococcus faecalis . Moreover, the phenotype of an insertionally inactivated degP allele in S. pyogenes is similar to that reported for E. coli , with temperature sensitivity for growth and enhanced sensitivity to reactive oxygen intermediates. Virulence studies in a mouse model of streptococcal infection indicate that a functional DegP protease is required for full virulence. These results suggest DegP as an attractive broad-spectrum target for future anti-infective drug development.

We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli . The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5′-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.

Type 6 streptococcal M protein produced by E. coli bearing plasmid pJRS42.13 (ColiM6) accumulates in the periplasmic space of this new host. No immunoreactive M protein was found either on the surface of the organism or in the culture medium. The ColiM6 protein was purified from the periplasm and the final preparation consisted of three protein bands of apparent molecular weight 55,000, 57,000, and 59,000. These three bands were identical in migration in SDS PAGE to that of the M protein present in freshly prepared crude periplasm. The amino acid composition of the ColiM6 protein was nearly identical to that of M protein isolated from streptococci with phage lysin (LysM6). Furthermore, except for the amino terminal residue of the LysM6 molecule, the amino terminal sequence of the ColiM6 molecule was identical to those of both LysM6 and M protein released from the streptococcus by limited peptic digestion (PepM6). These results reveal that the molecule produced in the E. coli and transported into the periplasm may be the complete M protein as it exists on the streptococcus. The results also indicate that the systems that process M protein for transport through the cytoplasmic membrane are similar in the streptococcus and E. coli. The purified ColiM6 protein was able to remove opsonic antibodies from both human and rabbit serum, as well as to stimulate the production of opsonic antibodies in rabbits, indicating that the immunodeterminants on this molecule are the same as those found on streptococcal-derived M molecules.