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Background Military personnel have an elevated risk of sustaining mild traumatic brain injuries (mTBI) and postconcussion symptoms (PCS). Smartphone apps that provide psychoeducation may assist those with mTBI or PCS to overcome unique barriers that military personnel experience with stigma and access to health care resources. Objective This study aims to (1) use the Mobile Application Rating Scale (MARS) to evaluate smartphone apps purporting to provide psychoeducation for those who have sustained an mTBI or a PCS; (2) explore the relevance, utility, and effectiveness of these apps in facilitating symptom management and overall recovery from mTBI and PCS among military personnel; and (3) discuss considerations pertinent to health care professionals and patients with mTBI when considering the use of mobile health (mHealth), including apps for mTBI psychoeducation. Methods A five-step systematic search for smartphone apps for military members with mTBI or PCS was conducted on January 31, 2020. Cost-free apps meeting the inclusion criteria were evaluated using the MARS and compared with evidence-based best practice management protocols for mTBI and PCS. Results The search yielded a total of 347 smartphone apps. After applying the inclusion and exclusion criteria, 13 apps were subjected to evaluation. Two apps were endorsed by the US Department of Veterans Affairs and the US Department of Defense; all the others (n=11) were developed for civilians. When compared with evidence-based best practice resources, the apps provided various levels of psychoeducational content. There are multiple considerations that health care professionals and those who sustain an mTBI or a PCS have to consider when choosing to use mHealth and selecting a specific app for mTBI psychoeducation. These may include factors such as the app platform, developer, internet requirement, cost, frequency of updates, language, additional features, acknowledgment of mental health, accessibility, military specificity, and privacy and security of data. Conclusions Psychoeducational interventions have a good evidence base as a treatment for mTBI and PCS. The use of apps for this purpose may be clinically effective, cost-effective, confidential, user friendly, and accessible. However, more research is needed to explore the effectiveness, usability, safety, security, and accessibility of apps designed for mTBI management.

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.

ST-246 is being evaluated as a treatment for pathogenic orthopoxvirus infections in humans. To this end, a phase 2, double-blind, randomized, placebo-controlled, multicenter trial was conducted to assess the safety, tolerability, and pharmacokinetics (PK) of ST-246 when administered as a single daily oral dose (400 mg or 600 mg) for 14 days in fed adult volunteers. ST-246 was safe and well tolerated, with no deaths or serious adverse events reported during the study. There was a low incidence of treatment-emergent adverse events (TEAEs), the most common of which were mild nausea and headache. There were no clinically significant results from laboratory assessments, vital sign measurements, physical examinations, or electrocardiograms. The PK and dose proportionality of ST-246 were determined. The PK analysis showed that steady state was achieved by day 5 for the ST-246 400-mg treatment group and by day 6 for the 600-mg group. The dose proportionality analysis showed that the 400- and 600-mg ratio of dose-normalized peak drug concentration in plasma ( C max ) and relative exposure for each dosing interval (AUC τ ) ranged from 80% to 85%. However, the 90% confidence intervals did not include 1.0, so dose proportionality could not be concluded. Overall, ST-246 was shown to be safe, and the PK was predictable. These results support further testing of ST-246 in a multicenter pivotal clinical safety study for licensure application.

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.

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.

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.

ST-246, a novel compound that inhibits egress of orthopoxvirus from mammalian cells, is being tested as a treatment for pathogenic orthopoxvirus infections in humans. This phase I, double-blind, randomized, crossover, exploratory study was conducted to compare the pharmacokinetics (PK) of a single daily 400-mg oral dose of ST-246 polymorph form I versus polymorph form V administered to fed, healthy human volunteers. Both forms appeared to be well tolerated, with no serious adverse events. The order of administration of the two forms had no effect on the results of the PK analyses. Form I and form V both exhibited comparable plasma concentration versus time profiles, but complete bioequivalence between the two forms was not found. Maximum drug concentration ( C max ) met the bioequivalence criteria, as the 90% confidence interval (CI) was 80.6 to 96.9%. However, the area under the concentration-time curve from time zero to time t (AUC 0- t ) and AUC 0-∞ did not meet the bioequivalence criteria (CIs of 67.8 to 91.0% and 73.9 to 104.7%, respectively). The extent of absorption of form I, as defined by AUC 0-∞ , was 11.7% lower than that of form V. Since ST-246 form I is more thermostable than form V, form I was selected for further development and use in all future studies.

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.

ST-246, a novel compound that inhibits egress of orthopoxvirus from infected cells, is being evaluated as a treatment for pathogenic orthopoxvirus infections in humans. This phase I, double-blind, randomized, placebo-controlled, escalating multiple-dose study was conducted to determine the safety, tolerability, and pharmacokinetics of ST-246 administered as a single daily oral dose of 250, 400, or 800 mg for 21 days to nonfasting healthy human volunteers. ST-246 appeared to be well tolerated, with no serious adverse events (AEs). Headache, for which one subject in the 800-mg group discontinued the study, was the most commonly reported AE in all treatment groups. The multiple-dose pharmacokinetics of ST-246 was well characterized. The day 21 mean elimination half-lives were calculated at 18.8, 19.8, and 20.7 h for each of the 250-, 400-, and 800-mg/day dose groups, respectively. Steady state was reached by day 6 (within 3 to 5 half-lives), saturable absorption was observed at the 800-mg dose level, and the fraction of parent drug excreted in the urine was very low. Based on these results, administration of 400 mg/day ST-246 can be expected to provide plasma concentrations above the efficacious concentration demonstrated in nonhuman primate models in earlier studies.

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.

T protein is a trypsin- and pepsin-resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize HindIII digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE6(1-299), coding for the amino-terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy-terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy-terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction-fragment size heterogeneity. Clearly, there is considerable diversity among T-serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.

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.

An active area of research in the development of Streptococcus gordonii for use as a bacterial commensal vector involves the identification and utilization of strong promoters for high-level expression of heterologous products. Escherichia coli plasmid vectors containing different streptococcal promoters often fail to become established in E. coli for unknown reasons. Therefore, it is desirable at times to transform S. gordonii, which is naturally competent, with small quantities of nascently ligated DNA without using E. coli first to amplify or screen the product. By comparing the efficiency of two methods used to induce competence in S. gordonii, it was shown that the use of a synthetic competence stimulating peptide substantially enhanced plasmid uptake by S. gordonii. We amplified the amylase-binding protein (abpA) promoter from the S. gordonii genome and, using a synthetic peptide to induce competence, directly introduced plasmid DNA containing this promoter into S. gordonii as an unamplified product of ligation. This plasmid facilitated abundant secretion of a heterologous product by S. gordonii. By assessing the levels of heterologous product secreted by two plasmid constructs, it was possible to evaluate the relative strength of two native promoters.

Streptococcus gordonii shows promise as a live mucosal vaccine vector for immunization against respiratory pathogens. In preparation for clinical trials to evaluate S. gordonii engineered to express group A streptococcal M protein antigens, we characterized the responses of 150 healthy volunteers to combined nasal and oral inoculation with approximately 1.5 × 10 9 CFU of SP204(1-1), an S. gordonii strain not bearing vaccine antigens. SP204(1-1) was selected for resistance to streptomycin and 5-fluoro-2-deoxyuridine to distinguish it from indigenous flora. In two antibiotic treatment studies, we performed serial culturing of nose, mouth, and saliva samples from 120 subjects treated with azithromycin beginning 5 days after inoculation to determine whether SP204(1-1) could be rapidly eliminated should safety concerns arise. A natural history study was performed to assess the time until spontaneous eradication in the remaining 30 subjects, who did not receive the antibiotic and who were monitored with repeated culturing for 14 weeks after inoculation. SP204(1-1) was generally well tolerated. Symptoms reported most often within 5 days of inoculation were nasal congestion (36%), headache (30%), and sore throat (19%). The strain was detected by culturing in 98% of subjects. A single dose of azithromycin eliminated colonization in 95% of subjects; all subjects receiving a 5-day course of an antibiotic showed clearance by day 11. Without the antibiotic, 82% of subjects showed spontaneous eradication of the implanted strain within 7 days, and all showed clearance by 35 days. The results of these clinical trials provide encouragement that the use of S. gordonii as a live mucosal vaccine vector is a feasible strategy.

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.

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.

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.

ST-246 is a novel, potent orthopoxvirus egress inhibitor that is being developed to treat pathogenic orthopoxvirus infections of humans. This phase I, double-blind, randomized, placebo-controlled single ascending dose study (first time with humans) was conducted to determine the safety, tolerability, and pharmacokinetics of ST-246 in healthy human volunteers. ST-246 was administered in single oral doses of 500, 1,000, and 2,000 mg to fasting healthy volunteers and 1,000 mg to nonfasting healthy volunteers. ST-246 was generally well tolerated with no serious adverse events, and no subject was withdrawn from the study due to ST-246. The most commonly reported drug-related adverse event was neutropenia, which was found, upon further analysis, not to be treatment related. ST-246 was readily absorbed following oral administration with mean times to maximum concentration from 2 h to 3 h. Absorption was greater in nonfasting volunteers than in fasting volunteers. Administration of ST-246 resulted in exposure levels predicted to be sufficient for inhibiting orthopoxvirus replication compared to exposure levels in nonhuman primates in which ST-246 protected animals from lethal orthopoxvirus infection.

In studies primarily confined to the amino-terminal region of the fibrillar group A streptococcal M protein, only limited immunological crossreactions have been observed among M serotypes. In this investigation, two monoclonal antibodies generated against nearly the entire M6 molecule (LysM6) were used to determine the extent of crossreactions among M serotyping strains and to localize their epitopes on the M molecule. Colony blot and immunoblot analyses revealed that an epitope responsible for crossreactions among 5 of the 56 strains of different M serotypes tested is located in the amino-terminal half of the molecule, distal to the cell surface. In contrast, a more common crossreactive epitope, reacting with 20 of the 56 strains, is located near the middle of the M molecule. These studies also reveal that the more conserved determinant, located more proximally to the cell surface, is accessible to the immune system, even on the whole organism, and, thus, may be useful in devising means to protect against infections by multiple group A streptococcal M serotypes.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.

The antigenic relatedness of surface-exposed portions of M protein molecules derived from group A streptococcal isolates representing more than 50 distinct serotypes was examined. The data indicate that the majority of serotypes fall into two major classes. Class I M protein molecules share a surface-exposed, antigenic domain comprising the C repeat region defined for M6 protein. The C repeat region of M6 protein is located adjacent to the COOH-terminal side of the pepsin-susceptible site. In contrast, Class I M proteins display considerably less antigenic relatedness to the B repeat region of M6 protein, which lies immediately NH2-terminal to the pepsin site. Surface-exposed portions of Class II M proteins lack antigenic epitopes that define the Class I molecules. Studies in the 1970s demonstrated that M protein serotypes can be divided into two groups based on both immunoreactivity directed to an unknown surface antigen (termed M-associated protein) and production of serum opacity factor. These two groups closely parallel our current definition of Class I and Class II serotypes. Both classes retain the antiphagocytic property characteristic of M protein, and Class II M proteins share some immunodeterminants with Class I M proteins, although the shared determinants do not appear to be exposed on the streptococcal surface. Nearly all streptococcal serotypes associated with outbreaks of acute rheumatic fever express M protein of a Class I serotype. Thus, the surface-exposed, conserved C repeat domain of Class I serotypes may be a virulence determinant for rheumatic fever.

One of 19 mAbs against the native group A streptococcal M6 protein proved opsonic for type 6 organisms in a bactericidal assay. The opsonic and three nonopsonic antibodies were selected for isotype and complement fixation studies based on previous knowledge of their epitope site on the M6 molecule. While mAb 3B8 (IgG3), whose epitope is in the NH2-terminal hypervariable region of the molecule (distal from the cell), and mAbs 10B6 (IgG2a) and 10F5 (IgG2b), both located in the conserved central region of the molecule, all fix complement, 10A11 (IgG1) did not. Only mAb 3B8 was opsonic despite the fact that mAbs 10B6 and 10F5 both exhibited similar complement-fixing capacity, binding titer, and surface exposure of epitopes. Analysis of antibodies raised against synthetic peptides representing various regions of the M6 protein showed that only the amino-terminal peptide (residues 1-21) was capable of eliciting opsonic antibodies, despite the fact that peptides from other areas produced antibodies with high-binding titers to the native M6 protein and also with the ability to bind to intact streptococcal cells. These results not only support the observed type specificity of opsonic antibodies, but also clearly point to the importance of the location of antibody binding on the M molecule relative to the actual functional capacity of the antibody with respect to the opsonization and phagocytosis of M6 streptococci. These results may underscore the recently observed role of complement Factor H in the antiphagocytic activity of the M protein.

Streptococcal M protein, the antiphagocytic molecule on the surface of the organism, was previously found to exhibit extensive size heterogeneity between as well as within M serotypes. In this study, methods were devised to isolate M protein size mutants within a laboratory-grown culture. We were able to isolate three independent M protein deletion mutants and one additional mutant, which was derived from the first deletion mutant. We found that these deletion mutants occur at a frequency of approximately 1 in 2 X 10(3) CFUs in culture. Functional studies revealed that the deletion mutants were able to survive as well as the parental strain in human blood. They also had the determinants necessary to absorb opsonic antibodies as well as the parent. Pepsin digestion experiments localized the deletions within the N-terminal half of the M molecule, which is distal to the cell wall surface. This is the region of the molecule in which extensive sequence repeats are found. This is consistent with the suggestion that the size changes may be the result of homologous recombination between the repeat regions in the gene. These results support the idea that strains showing M protein size variation within successive clinical isolates from single patients may be derived from the initial infecting organisms, and are not the result of separate unrelated acquisitions of the same serotype. This size change may be important in the survival of the streptococcus in vivo.

mAbs 10A11, 10B6, and 10F5, raised against the native group A streptococcal M6 protein, were examined for their crossreactivity with non-laboratory passaged clinical isolates, representing 58 M serotypes, by bacterial dot blot immunoassay. mAb 10A11 crossreacted with 9, mAb 10B6 with 30, and mAb 10F5 with 30 different non-M6 serotypes. To identify the epitopes for these antibodies, the native M6 protein was cleaved with pepsin or staphylococcal V8 protease. Resultant peptides were purified by HPLC, examined for binding to crossreactive mAbs in ELISA, and reactive peptides were subjected to amino acid sequence analysis. Peptides were aligned with the amino acid sequence of the entire M6 protein predicted by the DNA sequence of the M6 gene. Competitive inhibition studies using peptides synthesized on the basis of peptide and DNA sequences, in concert with selective blocking of amino acid residues, allowed for the further identification and placement of these crossreactive epitopes within the M6 molecule. The 10A11 epitope was located within the six amino acid residues at position 134-139, which repeat at positions 159-164 and 184-189 within the variable amino terminal half of the native molecule. The conserved 10B6 and 10F5 epitopes were positioned within a 15-amino-acid span at position 275-289, with the possibility that either epitope could have been repeated at residues 239-247. Chemical modification of amino acids within this sequence aided in the differentiation of these two epitopes. Such studies should aid in the recognition of a sequence(s) common to a greater number of M serotypes, which may be useful for future vaccine development or group A streptococcal identification.

Fifty-three isolates of Streptococcus uberis from various sources were examined for the presence of a common antigen. Initially, a serum was produced in rabbits which, by using rocket line immunoelectrophoresis, proved to react with identity to all of the S. uberis crude extracts as well as group B and E streptococcal extracts. The antigen(s) responsible for this cross-reactivity was partially purified by Sephacryl S-200 gel chromatography and analyzed by fused rocket immunoelectrophoresis. Further analysis by immunodiffusion showed that probably two antigens in the gel chromatography-consolidated fractions were common to the S. uberis and group B and E isolates, but that one of the antigens present was unique to S. uberis. Trypsin destroyed the immunoreactivity of this antigen. Isolation of this common antigen could possibly alleviate some of the tedium associated with the identification of this organism.

Deletions of highly, but not entirely, homologous intragenic sequence repeats result in amino acid sequence and conformational changes in the M proteins of spontaneous M protein-size variants of group A streptococci. To determine if antigenic changes occurred as a result of these deletion mutations, monoclonal and polyclonal antibodies with defined epitopes were used in competition assays. Competing antigens were either purified pepsin-derived fragments (representing the amino-terminal half of the molecule) of parent and mutant M proteins or were intact bacterial cells. These assays showed that antigenic variation occurred at the site(s) of these deletions but not at adjacent or distant epitopes. Once cleaved from the bacterium by pepsin, the M molecules also underwent conformational changes, which were reflected in their ability to compete. A monoclonal antibody opsonic for M6 streptococci lost its ability to completely opsonize one of the size mutants in this study. Therefore, spontaneous intragenic events between repeats within emm-6, the structural gene for the M6 protein, do result in structural variations within the mutant M molecules. This variation alters the ability of certain antibodies, originally produced in response to sequences in the parental M molecule, to bind to the mutant M molecules or opsonize the mutant organisms. Group A streptococci have evolved a mechanism for generating antigenic diversity that differs from currently known mechanisms in other bacterial species.

The ability of the group A streptococcus to cause human infection is primarily attributable to the M protein, a molecule located on the surface of the cell wall. This protein enables the organism to resist phagocytic attack by human neutrophils, thus allowing the streptococcus to persist in the infected tissue until such time as antibodies are produced against the M molecule (1). Because type-specific antibodies to the M protein lead to the effective clearance of the invading streptococcus, the ability of the organism to vary the antigenic structure of the M molecule has probably been responsible for the survival of the streptococcus in nature (1,2). Lancefield showed that, though delayed in their appearance, once formed, type-specific opsonic antibodies persist in the host for more than 30 years (2). To date, more than 80 se- rologically distinct M molecules have been identified in addition to a large number of clinical isolates for which typing sera are not yet available ((3), WHO meeting, Tokyo, Japan 1985).

Streptococcal M protein is a coiled-coil fibrillar structure extending about 60 nm from the cell wall. From DNA sequencing of the M6 protein gene, it has been determined that the C-terminal end contains a membrane anchor and an adjacent cell wall stabilization domain, both of which are similar to C-terminal regions of surface proteins in other gram-positive organisms. Studies with monoclonal antibodies revealed that the C-terminal half of the protein is conserved among M proteins of different serotypes, whereas the N-terminal half varies. The M protein contains tandem repeats, which, through homologous recombination, are responsible for the observed size variation of the M proteins from different streptococcal strains. M protein size mutants occur in a laboratory-grown culture at a frequency of 1/2,000 colony-forming units. DNA sequence analysis of the M gene in size mutants derived from a single strain suggests that sequence changes which result from homologous recombination may play a role in the antigenic variation of M protein.

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.