Adenovirus Microsatellite Reveals Dynamics of Transmission during a Recent Epidemic of Human Adenovirus Serotype 14 Infection

This study reveals diverse-length polymorphisms in long mononucleotide repeats (microsatellites) in several serotypes of epidemic human respiratory adenovirus. The length of one of these microsatellites, a homopolymeric thymidine [poly(T)] repeat, is measured in 68 isolates of adenovirus serotype 14. These isolates were collected during a series of sudden and sometimes fatal outbreaks among both military recruits and civilians as the virus emerged for the first time in the United States in 2006 and 2007. The results demonstrate the usefulness of adenoviral microsatellites as high-resolution molecular strain markers. The described homopolymer is hypervariable in length, varying from 12 to 17 bp in the analyzed sample set. All intermediate lengths were identified in at least one isolate. Furthermore, the specific length of the marker is stable for significant periods of time (up to 7 months) at individual sites where the virus is in consistent circulation. The microsatellite also can maintain specific length identity through site-to-site transmission events, as determined by the analysis of isolates from three advanced training sites that appeared to be subject to pathogen transfer from one of the affected recruit training installations. Public database searches revealed that the polymorphic nature of the microsatellite extends to other species B serotypes, and that other polymorphic microsatellites can be identified readily in a variety of epidemic respiratory adenovirus clades. This study shows that microsatellites are a ubiquitous source of polymorphic markers for human adenoviruses and demonstrates their use through an epidemiological analysis of isolates from a recent North American epidemic.Adult respiratory adenoviruses cause symptoms ranging from, commonly, febrile respiratory illness (FRI) to, more rarely, pneumonia and death (2, 21, 24). In civilian populations, they occur both sporadically and in local and national epidemics. Epidemics often are associated with the emergence of new variants (genome types) of otherwise common serotypes (8, 19) or the reemergence of recently rare serotypes (4, 11, 16).Adenoviruses of three species, human adenovirus serotypes B, C, and E (HAdV-B, HAdV-C, and HAdV-E, respectively), are strongly associated with respiratory disease in otherwise healthy people (2, 21, 22, 24). HAdV-C serotypes include HAdV-1, HAdV-2, HAdV-5, and HAdV-6 and are endemic among children and young adults (20). Most adults are immune to these serotypes. The sole HAdV-E serotype, HAdV-4, and several HAdV-B serotypes, including HAdV-3, HAdV-7, and HAdV-21, are commonly associated with epidemic outbreaks of FRI and pneumonia in healthy adults and children throughout the world (16, 17, 24, 21). Two other HAdV-B serotypes, HAdV-11a and HAdV-14, had, until recently, been identified only in association with respiratory disease epidemics in rare (though severe) outbreaks in Eurasia (3, 5, 16). Prior to the turn of the century, HAdV-14 had never been isolated from any case in North or South America (16).During 2006 and 2007, HAdV-14 was identified in a series of outbreaks across the United States, in association with widespread FRI and at least 10 documented pneumonia fatalities (4, 12, 16). This phenomenon was tracked simultaneously both in civilian populations, by the Centers for Disease Control and Prevention and local public health agencies (4), and in military recruits, by U.S. Department of Defense public health agencies (16). Detailed comparisons of collected strains, including the sequencing of fiber and hexon genes and whole-genome restriction analysis (genome typing), revealed that all of these events were caused by a single, apparently homogeneous, strain (12). The identified strain (genome type HAdV-14a) was significantly diverged from the prototypical Eurasian strain (HAdV-14p) identified in outbreaks in the mid-1900s (12).The full-genome sequencing of multiple HAdV-14a isolates, including both a fatal pneumonia isolate from a severe outbreak in Texas in 2007 and an isolate from a mild outbreak in California in 2006, which had little effect on local adenoviral illness rates, revealed only two genetic polymorphisms between the two strains. Both polymorphisms were noncoding. One of these was a synonymous base mutation in the fiber gene (H. S. Houng, unpublished data). This high degree of homogeneity (clonality) did not offer a simple way to track different lineages of the emerging virus or suggest a functional genetic etiology of the observed variation in clinical impact at different sites.The second polymorphism was an insertion/deletion mutation in a mononucleotide microsatellite repeat, consisting of 17 consecutive thymidines [T(17)] in one sequenced isolate and T(13) in the other. This polymorphism was identified in the noncoding region at the 3′ end of the terminal binding protein (TBP) gene. The repeat likely serves as the polyadenylation signal for the TBP mRNA. Microsatellite repeats, also referred to as variable-number tandem repeats, simple sequence repeats, and amplified fragment length polymorphisms, are widely recognized as extremely variable high-resolution strain markers (6, 7, 13, 15, 18, 26). These repetitive sequences experience an extremely high rate of replication slippage, resulting in length changes through the gain or loss of single or, more rarely, multiple repeat units (14, 23, 26). Microsatellites therefore vary by length rather than by sequence content, and resulting changes are readily reversed by further mutation (unlike random base substitutions) (14, 23, 26). For extremely clonal populations such as that of the emerging HAdV-14 strain, microsatellites and other hypervariable repeats can offer the only available source of variable genetic strain markers.The identified microsatellite locus was sequenced from 56 isolates collected during seven recent recruit training center outbreaks, representing 2 years of HAdV-14 surveillance following the initial emergence in the U.S. military. These recruit centers represent the first step in military training and host large numbers of new military recruits from across the United States. Nine duplicate isolates (split aliquots of the same isolate) were sequenced to measure repeatability, because microsatellites can mutate quickly enough to generate mixed populations. When site-specific microsatellite alleles were revealed, the analysis was extended to 11 isolates from three advanced U.S. Air Force training sites. These sites host military personnel for specialized training following initial recruit training. The advanced training sites studied here received large numbers of recruit trainees from Lackland Air Force Base (AFB) in San Antonio, TX, during the time of the most severe HAdV-14 outbreak documented among U.S. recruits, and subsequently suffered from HAdV-14 outbreaks themselves. It was hypothesized that isolates from these advanced Air Force training centers would reflect the microsatellite identity of the Lackland isolates and hence reflect the transmission that was thought to have occurred through trainee movement.In order to expand the potential set of microsatellite alleles available for use as strain markers in epidemiological studies, public databases were used to identify several other potentially polymorphic microsatellite repeats in respiratory adenoviruses. The lengths of newly identified microsatellites then were compared across all published sequences to identify polymorphisms. Those microsatellites exhibiting a high degree of polymorphism are reported here with the observed distribution of allele lengths.     

Sequence Analysis of Porcine Adenovirus Serotype 5 Fibre Gene: Evidence for Recombination

The nucleic acid and deduced amino acid sequence of the fibre gene of the HNF-61 strain of porcine adenovirus serotype 5 (PAdV-5) was determined and compared to that of the HNF-70 strain of the same serotype (Nagy et al., J Gen Virol 82, 525–529, 2001) and also to adenovirus fibre genes from the genera Mastadenovirus and Atadenovirus. The putative HNF-61 and HNF-70 proteins were similar to each other, with 90% amino acid identity. Conserved amino acid sequences described for mastadenovirus fibre shafts were identified in the shaft regions of both PAdV-5 fibres, except for the so-called TLWT motif. The head regions of the PAdV-5 fibre did not resemble any of the known mastadenovirus fibre heads, but they showed characteristics of the fibre head protein sequences of viruses grouped in the proposed genus Atadenovirus (Benkö et al., Virus Taxonomy, Seventh Report of the International Committee on the Taxonomy of Viruses, Academic Press, New York, San Diego, 2000, pp. 227–238). The findings suggested recombination between viruses of different adenovirus genera.     

Safety and Immunogenicity of the Merck Adenovirus Serotype 5 (MRKAd5) and MRKAd6 Human Immunodeficiency Virus Type 1 Trigene Vaccines Alone and in Combination in Healthy Adults

Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of ≤18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (≤200 or >200) and Ad6 titers (≤18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to ≥2 vaccine antigens were 14%, 63%, and 71% at 10⁹, 10¹⁰, and 10¹¹ viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 10¹⁰ vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.Adenovirus (Ad) vectors have been investigated as a vaccination strategy for inducing cell-mediated immunity (CMI) to several viral and bacterial pathogens (11, 13, 22, 24, 26). In preclinical and phase I studies, vaccination with attenuated Ad serotype 5 (Ad5) vectors expressing human immunodeficiency virus type 1 (HIV-1) gag elicited strong CMI responses in both macaques and humans (4, 5, 14, 20, 23). Although a similar Ad5-vectored trivalent HIV-1 vaccine did not prevent or modulate infection in the proof-of-concept STEP trial (2), adenoviruses remain attractive candidates as vectors for inducing CMI against a variety of common infections.Diminished immune responses to transgenes carried by Ad5 vectors as a result of preexisting Ad5-specific immunity have been a concern from the advent of Ad5-based vaccine trials in humans (2, 5, 13, 16, 18, 25). High preexisting titers of neutralizing antibodies against Ad5 substantially diminished CMI responses to HIV-1 vaccines using Ad5 vectors (2, 5, 16, 18). Most North American adults have demonstrable neutralizing antibody against Ad5, and nearly one-third have relatively high titers (21, 25, 26). The frequency and magnitude of Ad5 titers are even higher in other parts of the world (8, 21). Neutralizing antibody against Ad6 is present less frequently and in lower titers (8, 21). Relatively few individuals would be expected to have high titers of antibodies against both Ad5 and Ad6.Strategies for overcoming preexisting Ad5 immunity include increasing the dose of Ad5-based vaccines, employing heterologous prime-boost regimens, or using different vectors, such as alternative adenovirus serotypes (3, 15, 26). The current trial was designed to explore the use of Ad6 with or without Ad5 as a vaccine vector for delivering HIV-1 gag, nef, and pol transgenes.(These data have been presented in part at the AIDS Vaccine 2007 Conference, Seattle, WA, August 2007 [12a, 12b].)     

Intramuscular Delivery of Adenovirus Serotype 5 Vector Expressing Humanized Protective Antigen Induces Rapid Protection against Anthrax That May Bypass Intranasally Originated Preexisting Adenovirus Immunity

Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a single dose of 10⁸ infectious units of Ad5-PAopt achieved 100% protection from challenge with 10 times the 50% lethal dose (LD₅₀) of anthrax lethal toxin 7 days after vaccination. Although preexisting intranasally induced immunity to Ad5 slightly weakened the humoral and cellular immune responses to Ad5-PAopt via intramuscular inoculation, 100% protection was achieved 15 days after vaccination in Fisher 344 rats. The protective efficacy conferred by intramuscular vaccination in the presence of preexisting intranasally induced immunity was significantly better than that of intranasal delivery of Ad5-PAopt and intramuscular injection with recombinant PA and aluminum adjuvant without preexisting immunity. As natural Ad5 infection often occurs via the mucosal route, the work here largely illuminates that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficient approach for protecting against emerging anthrax.     

Assessment of Chimpanzee Adenovirus Serotype 63 Neutralizing Antibodies Prior to Evaluation of a Candidate Malaria Vaccine Regimen Based on Viral Vectors

Prior to a chimpanzee adenovirus-based (ChAd63) malarial vaccine trial, sera were collected to assess ChAd63-specific neutralizing antibody titers in Banfora (Burkina Faso). The low neutralizing antibody titers reported in both adults and children (median titers, 139.1 and 35.0, respectively) are encouraging for the potential use of ChAd63 as a malarial vaccine vector.     

Outbreak of Acute Respiratory Infection among Infants in Lisbon,Portugal, Caused by Human Adenovirus Serotype 3 and a New 7/3 Recombinant Strain

Human adenoviruses (AdVs) typically cause mild illnesses in otherwise healthy hosts. We investigated a pediatric outbreak of acute respiratory infection with fatal outcomes that occurred in Lisbon, Portugal, in 2004. Biological specimens were collected from 83 children attending two nurseries, a kinesiotherapy clinic, and the household of a nanny. Adenovirus infection was confirmed in 48 children by PCR and virus isolation. Most (96%) isolates were classified as being of subspecies B1. Phylogenetic analysis of fiber and hexon gene sequences revealed that most infants were infected with AdV serotype 3 (AdV3) strains. Infants attending one nursery harbored a new recombinant strain containing an AdV serotype 7 hexon and serotype 3 fiber (AdV7/3). Both the AdV3 and the AdV7/3 strains caused fatal infections. Two different serotype 3 strains were circulating in Lisbon in 2004, and the new AdV7/3 recombinant type originated from only one of those strains. These results demonstrate that recombination leads to the emergence of new adenovirus strains with epidemic and lethal potential.Human adenoviruses (HAdVs) have been associated with a wide spectrum of clinical diseases with respiratory and gastrointestinal symptoms (23, 32). Severe illness can occur in newborns, elderly individuals, and patients with underlying medical conditions. In otherwise healthy adults, infections caused by human adenoviruses do not represent a life-threatening clinical condition. Adenoviruses are characterized by a linear double-stranded DNA genome of 2 to 45 kbp that encodes 30 to 40 proteins (6). HAdV comprises 51 serotypes (HAdV-1 to HAdV-51), on the basis of type-specific antiserum-mediated neutralization of infectivity (10). The serotypes can be divided into seven species, named HAdV-A to HAdV-G, on the basis of hemagglutination inhibition and biochemical criteria (13). HAdV-B is further classified into subspecies B1 and B2, which use different cellular receptors for viral entry (29). These variants can be segregated by different geographic areas, time periods, and clinical conditions.Serotype identification is critical for epidemiological surveillance, the detection of new strains, assessment of treatment efficacy, and understanding the pathogenesis of HAdV. For example, acute respiratory disease is primarily caused by HAdV-B1 serotypes 3, 7, 16, and 21; HAdV-B2 serotypes 11 and 14; and HAdV-E serotype 4 (8, 23, 25, 28, 35, 38, 41). Respiratory infections caused by HAdV-B1 serotypes 3 and 7 (16) and HAdV-B2 serotype 14 (17) are potentially fatal. Neutralization tests are the classical reference method used for the typing of adenovirus and require virus isolation from infected organs or tissues (20). The main type-specific neutralizing epitope, the ɛ determinant, consists of loop 1 (L1) and loop 2 (L2) on the hexon protein, the major capsid protein and the most abundant structural protein (26). Cases of the failure of neutralization with the available antisera require extensive cross-neutralization studies to define a new HAdV type. To circumvent the practical problems associated with traditional serum neutralization studies, molecular methods for the typing of adenovirus have been established. Examples are restriction fragment length polymorphism (RFLP) analysis of adenoviral DNA (16), PCR-based assays (1, 36), and microarray-based methods (36). However, these methods cannot discriminate between all serotypes and do not allow detailed studies of molecular epidemiology and viral evolution to be performed.More recently, analysis of the nucleotide and amino acid sequences from different genes has shown that adenovirus species form three distinct phylogenetic clusters: HAdV-C belongs to cluster 1; HAdV-A and HAdV-F belong to cluster 2; and HAdV-B, HAdV-D, and HAdV-E belong to cluster 3 (6, 22). In addition, phylogenetic analysis of selected gene fragments has increasingly been used to classify human adenoviruses at the serotype and species levels (7, 19, 40), to detect cases of coinfection with multiple adenoviral species (36, 38), and to identify new recombinant strains formed between similar species (18, 37, 41) or different species (19). Finally, phylogenetic analysis has become an important tool in the epidemiological investigation of many disease outbreaks caused by adenovirus (11, 17, 27, 40, 41). In the present study, we have used epidemiological, virological, and molecular phylogenetic methods to investigate the causes and origin of a recent outbreak of acute respiratory infection in Lisbon, Portugal, that resulted in the deaths of two children.     

Necrotizing Fasciitis Caused by Haemophilus influenzae Serotype f

Haemophilus influenzae is a rare cause of soft tissue infection. In this report, we present a case of multifocal necrotizing fasciitis in a healthy adult patient, secondary to Haemophilus influenzae serotype f infection, and we review literature on this rare cause of necrotizing fasciitis.     

The Th1 Immune Response to Plasmodium falciparum Circumsporozoite Protein Is Boosted by Adenovirus Vectors 35 and 26 with a Homologous Insert

The most advanced malaria vaccine, RTS,S, is comprised of an adjuvant portion of the Plasmodium falciparum circumsporozoite (CS) protein fused to and admixed with the hepatitis B virus surface antigen. This vaccine confers short-term protection against malaria infection, with an efficacy of about 50%, and induces particularly B-cell and CD4⁺ T-cell responses. In the present study, we tested by the hypothesis that the Th1 immune response to CS protein, in particular the CD8⁺ T-cell response, which is needed for strong and lasting malaria immunity, is boosted to sustainable levels vectors adenovirus and 26 with an homologous insert 35 (Ad35.CS/Ad26.CS). In this study, we evaluated immune responses induced with vaccination regimens based on an adjuvant-containing, yeast-produced complete CS protein followed by two recombinant low-seroprevalence adenoviruses expressing P. falciparum CS antigen, Ad35.CS (subgroup B) and Ad26.CS (subgroup D). Our results show that (i) the yeast (Hansenula polymorpha)produced, adjuvanted full-length CS protein is highly potent in inducing high CS-specific humoral responses in mice but produces poor T-cell responses, (ii) the Ad35.CS vector boosts the gamma interferon-positive (IFN-γ⁺) CD8⁺ T-cell response induced by the CS protein immunization and shifts the immune response toward the Th1 type, and (iii) a three-component heterologous vaccination comprised of a CS protein prime followed by boosts with Ad35.CS and Ad26.CS elicits an even more robust and sustainable IFN-γ⁺ CD8⁺ T-cell response than one- or two-component regimens. The Ad35.CS/Ad26.CS combination boosted particularly the IFN-γ⁺ and tumor necrosis factor alpha-positive (TNF-α⁺) T cells, confirming the shift of the immune response from the Th2 type to the Th1 type. These results support the notion of first immunizations of infants with an adjuvanted CS protein vaccine, followed by a booster Ad35.CS/Ad26.CS vaccine at a later age, to induce lasting protection against malaria for which the Th1 response and immune memory is required.Almost 40 years after the feasibility of vaccination against malaria was first demonstrated by means of irradiated sporozoites (9), a vaccine modality that efficiently induces long-lived protective immunity remains elusive. The most advanced circumsporozoite (CS)-based malaria vaccine candidate to date is RTS,S, a vaccine based on a fragment of Plasmodium falciparum circumsporozoite (CS) protein fused to and admixed with hepatitis B virus surface protein. In adults, RTS,S with the adjuvant AS02 has consistently conferred 40% protection against malaria infection upon sporozoite challenge (54). Even though RTS,S/AS02 induces high-level CS-specific antibody responses, the induced T-cell responses are weak (21). As the Th1 response, particularly gamma interferon (IFN-γ) and CD8⁺ T cells, is associated with protection, novel adjuvant systems were developed with the aim of improving the induced T-cell response while maintaining potent levels of CS-specific antibody responses. One of these novel adjuvant systems, AS01, demonstrated its suitability in mice, as it improved CS-specific CD4⁺ T-cell responses and led to induction of CD8⁺ T cells (32). Nonhuman primate studies also demonstrated that RTS,S with AS01 adjuvant induces strong CS-specific antibody responses as well as mean higher frequencies of IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing CD4⁺ T cells than those generated by RTS,S with AS02 adjuvant. However, the induction of CD8⁺ T cells was not confirmed in this nonhuman primate study (32). In humans, RTS,S/AS01 has been shown to induce high titers of CS-specific antibodies and higher numbers of Th1 CD4⁺ T cells than those generated by RTS,S/AS02 but no CS-specific CD8⁺ T cells (22). However, RTS,S/AS01 was able to afford 50% protection against malaria infection in adults upon sporozoite challenge (22) and 53% efficacy against disease in children between the ages of 5 and 17 months (5). These results, albeit far from being optimal, supported the progress of RTS,S/AS01 to phase III clinical trial testing in early 2009, and these trials enrolled children at the age of 6 weeks to 17 months at multiple sites in sub-Saharan Africa. It is anticipated that RTS,S/AS01 will be the first licensed malaria vaccine, provided its efficacy is confirmed in the phase III trial.Although our understanding about the correlate(s) of protection for malaria is limited, there is ample evidence that CS protein-specific antibodies, CD8⁺ T cells, and Th1 cytokines, particularly IFN-γ, play a central role in controlling the preerythrocytic and early liver stages of malaria (19, 20, 35, 47, 57). Adenovirus (Ad) vectors are particularly suited for induction of IFN-γ-producing CD8⁺ T cells required to combat malaria infection (33, 43), due to intracellular expression of a transgene inserted in the vector genome and efficient routing of expressed protein toward the class I presentation pathway. Recently, we demonstrated the advantage of utilizing two recombinant adenoviral vectors derived from distinct serotypes, Ad type 35 CS (Ad35.CS) and Ad5.CS, in a heterologous prime-boost regimen in mice and nonhuman primates (46). This heterologous prime-boost regimen elicited a high-level CS-specific IFN-γ⁺ T-cell response as well CS-specific Th1-type antibodies able to bind malaria parasites. Though the Ad5-based vectors are very potent vaccines, the high prevalence of preexisting immunity toward Ad5 in the human population hampers their immunogenicity and clinical utility (8, 38). The low seroprevalence of Ad5-neutralizing antibodies in infants of 6 months to 1.5 years of age offers an opportunity to administer Ad5-based vaccines to this population without antibodies interfering and neutralizing the vaccine efficacy (42); however, acceptance of this approach by regulatory agencies may remain difficult to obtain. Novel vaccine vectors based on rare low-seroprevalence Ad serotypes have an advantage of not being hampered by anti-Ad5 immunity while inducing a strong immune response (1, 4, 28, 33, 41).Within this study, we evaluated whether vaccination with Ad35.CS and Ad26.CS can enhance the CS-specific immune response induced by a yeast-produced full-length CS protein vaccine and, in particular, whether the combined vaccination sustainably potentiates the Th1 responses necessary for protection against malaria. The Ad35.CS vaccine candidate is currently being evaluated in a phase 1 clinical study, in partnership with the National Institute of Allergy and Infectious Diseases, and so far, it has been shown to be safe. Candidate Ad35-based vaccines against other infectious diseases, i.e., tuberculosis and HIV infection, have also been clinically evaluated and demonstrated to be safe and immunogenic. Recently, an Ad26 vector vaccine against HIV was also clinically assessed in a phase I study, which showed that a 3-dose regimen of this HIV candidate vaccine is safe and immunogenic. Based upon encouraging results, the clinical testing of the combination of Ad35- and Ad26-based vaccines against malaria and HIV is in preparation.     

Serotype Identification of Group B Streptococci by PCR and Sequencing

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (cps) gene clusters to further define partial cps gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.     

Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae

The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 10⁸-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 10⁷- to 10⁹-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.Otitis media remains a significant health problem for children in this country and elsewhere in the world (15, 16). Most children in the United States have had at least one episode of otitis by the third birthday, and one-third have had three or more episodes (51). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (50). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for prevention of otitis media in young children (26).Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of cases of acute otitis media (20, 53). Streptococcus pneumoniae has been the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (20, 53). Nontypeable Haemophilus influenzae (NTHi) is the second most common bacterium recovered and accounts for 20% to 30% of cases of acute otitis media and a larger percentage of cases of chronic and recurrent disease (37). Interestingly, since introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccination schedule, nontypeable Haemophilus influenzae has become an even more common cause of acute and recurrent middle ear disease, often surpassing Streptococcus pneumoniae in frequency of recovery from middle ear fluid specimens (12, 18).Many different antigens have been suggested as possible nontypeable Haemophilus influenzae vaccine candidates (1, 5, 23, 43, 44, 63). In our early work, we demonstrated that development of bactericidal antibody in the sera of children who had recovered from acute NTHi otitis media was associated with the appearance of serum antibodies directed against highly immunogenic high-molecular-weight (HMW) proteins (7). This work led subsequently to the identification and characterization of the HMW1 and HMW2 (HMW1/HMW2) family of proteins (8). The HMW1/HMW2 proteins have subsequently been shown to be major adhesins of nontypeable Haemophilus influenzae (57) as well as targets of opsonophagocytic (65, 66) and protective (6) antibodies. The HMW1/HMW2-like proteins are expressed by approximately 75% of NTHi strains (8, 58). The 25% of NTHi strains that do not express HMW1/HMW2-like proteins also express immunogenic high-molecular-weight proteins that are recognized by human convalescent-phase serum antibodies (11). Almost all the HMW1/HMW2-negative strains have subsequently been shown to express a second distinct class of adhesins known as Hia proteins (11). The Hia proteins are members of a large family of bacterial proteins known as autotransporters that are found in many Gram-negative bacteria (28, 69). The Hia proteins have also recently been shown to serve as targets for opsonophagocytic antibodies (64). Nearly all NTHi strains that lack HMW1/HMW2 proteins contain a hia gene and express a Hia protein, and conversely, strains that express HMW1/HMW2 proteins lack a hia gene (11, 58).Several groups have begun exploring mucosal and, in particular, nasopharyngeal immunization strategies to stimulate a protective immune response in the upper respiratory tract and middle ear (19, 24), and results to date have been encouraging (4, 29, 32, 47). Intranasal immunization has a number of potential advantages over traditional parenteral immunization approaches for prevention of otitis media. The presence of abundant microvilli in the nasal cavity greatly increases the available surface area of this anatomical site, thereby generating a large absorptive surface (34). Immunization via the nasal cavity also allows direct delivery of immunogens of interest to inductive/effector sites (i.e., nasal mucosa-associated lymphoid tissue [NALT] and Waldeyer''s ring), which can result in both a systemic and a mucosal response (14, 33, 62). Furthermore, the nasal cavity is readily accessible and allows for noninvasive delivery of antigens or vaccines, thus eliminating the need for trained staff or the use of sterile needles and syringes. The option of treating or immunizing against human disease with such a noninvasive technique would almost certainly result in increased patient compliance if intranasal vaccines advance to the clinic.A number of mucosal vaccination strategies continue to be actively explored (41, 49). Adenoviruses have been identified as promising live recombinant vaccine vectors based upon their ability to induce high levels of heterologous gene expression (13, 25, 55, 60) and to stimulate mucosal immunity in the upper respiratory tract, their natural site of replication. E1-deletion-containing replication-defective adenoviral recombinants based on human serotype 5 (Adhu5) have been tested as vaccine candidates for prevention of a number of infectious diseases (21, 60). Studies of adenoviral vaccines based upon the H5N1 hemagglutinin protein (30), surface proteins of Streptococcus pneumoniae (3), the rabies virus glycoprotein (68), the circumsporozoite protein of Plasmodium falciparum (54), and the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) (27) have demonstrated that E1-deletion-containing vaccines induce excellent B-cell and CD8⁺-T-cell responses in experimental animals, even if given at moderate doses.Recombinant adenovirus (rAd) vectors have yet to be investigated as potential vaccine candidates for prevention of otitis media. However, they are particularly attractive candidates for the reasons noted above. The objective of the present study was to construct E1-deletion-containing replication-defective recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia adhesion protein of nontypeable Haemophilus influenzae and to assess their immunogenicity in the chinchilla experimental model.     

Early Changes in the Serotype Distribution of Invasive Pneumococcal Isolates from Children after the Introduction of Extended-valent Pneumococcal Conjugate Vaccines in Korea, 2011-2013

This study was performed to measure early changes in the serotype distribution of pneumococci isolated from children with invasive disease during the 3-year period following the introduction of 10- and 13-valent pneumococcal conjugate vaccines (PCVs) in Korea. From January 2011 to December 2013 at 25 hospitals located throughout Korea, pneumococci were isolated among children who had invasive pneumococcal disease (IPD). Serotypes were determined using the Quellung reaction, and the change in serotype distribution was analyzed. Seventy-five cases of IPD were included. Eighty percent of patients were aged 3-59 months, and 32% had a comorbidity that increased the risk of pneumococcal infection. The most common serotypes were 19A (32.0%), 10A (8.0%), and 15C (6.7%). The PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F, and 6A) accounted for 14.7% of the total isolates and the PCV13 minus PCV7 types (1, 3, 5, 7F and 19A) accounted for 32.0% of the total isolates. Serotype 19A was the only serotype in the PCV13 minus PCV7 group. The proportion of serotype 19A showed decreasing tendency from 37.5% in 2011 to 22.2% in 2013 (P = 0.309), while the proportion of non-PCV13 types showed increasing tendency from 45.8% in 2011 to 72.2% in 2013 (P = 0.108). Shortly after the introduction of extended-valent PCVs in Korea, serotype 19A continued to be the most common serotype causing IPD in children. Subsequently, the proportion of 19A decreased, and non-vaccine serotypes emerged as an important cause of IPD. The impact of extended-valent vaccines must be continuously monitored.     

Attenuation of murine coronavirus infection by ammonium chloride

Ammonium chloride at a concentration of 20 mM delayed by 4-5 hr the production of virus progeny in mouse L-2 cells infected at high multiplicity with mouse hepatitis virus (MHV). This delay was seen in the production of both intracellular and extracellular virus. However, the final titers were similar to those produced by MHV-infected cells maintained in normal medium. The manifestation of virus-induced cell fusion was similarly found to be delayed, but not otherwise decreased in severity, when ammonium chloride was present in the culture medium. Ammonium chloride caused similar delays in production of virus-specific, positive-sense RNAs and of viral polypeptides. The relative proportions and apparent molecular weights of viral RNAs and polypeptides were similar to those found in MHV-infected cells cultured in normal medium. In vitro translation of endogenously produced viral RNAs in cell extracts, prepared from MHV-infected cells, was not inhibited by ammonium chloride. Thus, ammonium chloride has no specific, inhibitory effect on viral protein synthesis. Ammonium chloride did not reduce the number of virus-infected cells in culture, as monitored by infectious center assay. Analysis of early events in MHV infection showed that ammonium chloride did not affect adsorption or internalization of MHV by L-2 cells. However, the subsequent eclipse phase, as monitored by decline in infectivity of internalized virus inoculum proceeded less efficiently in the presence of ammonium chloride. On the basis of the known inhibitory effects of ammonium chloride on lysosomal/endosomal functions, the results suggest an endosomal mechanism of MHV uncoating. Thus the primary effect of ammonium chloride on MHV infection of L-2 cells is to attenuate virus uncoating, thereby chronologically displacing all subsequent virus-encoded functions.