Biomedical subjects

C G Hayes

Publications and source records attributed to C G Hayes.

At least 19 recordsLinked to original sources

PubMed2001

Synergistic neutralizing antibody response to a dengue virus type 2 DNA vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing GM-CSF.

We have previously shown that a dengue virus type 1 DNA vaccine expressing premembrane (prM) and envelope (E) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. In order to improve the immunogenicity of dengue DNA vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing GM-CSF on antibody responses. A dengue virus type 2 candidate vaccine containing prM and E genes was constructed in which the transmembrane and cytoplasmic regions of E were replaced by those of the lysosome-associated membrane protein (LAMP). The modified vaccine construct expressed antigen that was colocalized with endogenous LAMP in lysosomal vesicles of transfected cells, whereas the antigen expressed from the unmodified construct was not. It was hypothesized that targeting of antigen to the lysosomal compartment will increase antigen presentation by MHC class II, leading to stronger CD4-mediated immune responses. Mice immunized with the modified construct responded with significantly higher levels of virus neutralizing antibodies compared to those immunized with the unmodified construct. Coimmunization of mice with a plasmid expressing murine GM-CSF enhanced the antibody response obtained with either the unmodified or the modified construct alone. The highest antibody responses were noted when the modified construct was coinjected with plasmid expressing the GM-CSF gene. These results could form the basis for an effective tetravalent dengue virus DNA vaccine.

3T3 Cells↗

PubMed2001

West Nile virus: Uganda, 1937, to New York City, 1999.

West Nile virus, first isolated in 1937, is among the earliest arthropod-borne viruses discovered by humans. Its broad geographical distribution, not uncommon infection of humans, transmission by mosquitoes, and association with wild birds as enzootic hosts were well documented by the mid-1960s. However, West Nile virus was not considered to be a significant human pathogen because most infections appeared to result in asymptomatic or only mild febrile disease. Several epidemics had been documented prior to 1996, some involving hundreds to thousands of cases in mostly rural populations, but only a few cases of severe neurological disease had been reported. The occurrence between 1996 and 1999 of three major epidemics, in southern Romania, the Volga delta in southern Russia, and the northeastern United States, involving hundreds of cases of severe neurological disease and fatal infections was totally unexpected. These were the first epidemics reported in large urban populations. A significant factor that appeared in common to all three outbreaks was the apparent involvement of the common house mosquito, Culex pipiens, as a vector. This species had not previously been implicated as important in the transmission of West Nile virus. In addition the epidemic in the northeastern United States was unusual in the association of West Nile virus infection with fatal disease of birds, suggesting a change in the virulence of the virus toward this host. Understanding the risk factors that contributed to these three urban epidemics is important for minimizing the potential for future occurrences. This review will attempt to compare observations on the biology of West Nile virus made over about 60 years prior to the recent epidemics to observations made in association with these urban epidemics.

Animals↗

PubMed2001

Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Animals↗

PubMed2001

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Animals↗

PubMed2001

Short report: Antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine.

Recombinant proteins containing the B domain of dengue virus serotypes 1-4 fused to the maltose binding protein (MBP) of Escherichia coli were evaluated individually and as a tetravalent vaccine candidate in mice. Sera from mice immunized with monovalent DEN-MBP recombinant protein vaccines developed high titers of serotype homologous antibody in the enzyme-linked immunosorbent assay and the plaque-reduction neutralization test. Cross-reactive antibody titers were either several dilutions lower or not detectable. Sera from mice immunized with the tetravalent DEN subunit vaccine neutralized all 4 DEN viruses in the plaque-reduction neutralization test. The neutralizing antibody titers to each individual serotype were significantly greater than any cross-reactive neutralizing antibody titers induced by the monovalent vaccines, providing evidence that the tetravalent DEN recombinant subunit vaccine produced specific neutralizing antibody to all 4 serotypes of dengue virus.

ATP-Binding Cassette Transporters↗

PubMed2001

Haiti: absence of dengue hemorrhagic fever despite hyperendemic dengue virus transmission.

In 1994-1996, 185 strains of dengue (DEN) virus types 1, 2, and 4 were recovered from febrile United States and other United Nations military personnel in Haiti. We wondered whether risk factors for dengue hemorrhagic fever (DHF) existed and, if so, were DHF cases occurring among Haitian children. Dengue transmission rates were studied in 210 school children (6-13 years old) resident in Carrefour Borough, Port-au-Prince, Haiti. When sera were tested for plaque-reduction neutralizing antibodies to DEN 1-4 viruses, nearly 85% had antibodies to two or more DEN serotypes. The annual transmission rate was estimated at 30%, a rate observed in countries endemic for DHE Haitian DEN 2 isolates were genotype I, which are repeatedly associated with DHF cases in Southeast Asia and American regions. Despite positive virologic pre-conditions, DHF cases were not recorded by experienced Port-au-Prince pediatricians. These observations, which are reminiscent of those in Africa, provide further evidence of a dengue resistance gene in black populations.

Adolescent↗

PubMed2001

Characterization of antibody responses to combinations of a dengue-2 DNA and dengue-2 recombinant subunit vaccine.

A dengue-2 (DEN-2) DNA vaccine coding for the premembrane and envelope (E) proteins and a recombinant fusion protein containing the B domain of the DEN-2 E protein fused to the maltose-binding protein (MBP) of Escherichia coli both elicited neutralizing antibody in mice. In order to achieve more rapid protective immunity as well as to increase the persistence of neutralizing antibody, we primed mice with the DNA vaccine (D), the recombinant MBP protein (R), or both (RD) given simultaneously, and then boosted twice with either the R (R/R/R or D/R/R) or D (D/D/D or R/D/D) constructs alone or the RD (RD/RD/RD) combination. All of the recombinant protein vaccines were given with alum as an adjuvant. The serum antibody response measured by enzyme-linked immunosorbent assay was highest in D/D/D mice and RD/RD/RD mice. The D/R/R mice showed an intermediate response, and the R/D/D and R/R/R showed the lowest response. The geometric mean (GM) 50% neutralizationtiter (50% plaque reduction neutralization, or PRNT50) was marginally higher for RD/RD/RD mice (891) at 9 months after priming than that for R/R/R mice (724). T he lowest GM PRNT50 titers were seen in the D/D/D mice (33) and R/D/D mice (40), and the D/R/R group had a slightly higher titer (156) than these 2 groups. The predominant antibody subclass for the D/D/D mice was immunoglobulin (Ig) G2a, similar to mice infected with live virus. The R/R/R mice showed an exclusive IgGI antibody response, and the RD/RD/RD response also was predominantly IgGI. The antibody subclass pattern of the R/D/D and D/R/R mice showed a more balanced distribution of both IgG1 and IgG2a. Investigating the neutralizing capacity of antibody subclasses suggested that both IgG1 and IgG2a could neutralize DEN-2 virus. Our observations indicate that the combination RD prime-boost regimen warrants further investigation as a vaccine strategy to prevent dengue infection.

Animals↗

PubMed2000

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the naïve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.

Animals↗

PubMed2000

Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein.

Recombinant plasmid DNA constructs expressing truncated or full-length dengue-1 envelope (E) with or without the pre-membrane (prM) were tested for immunogenicity in mice, as candidate dengue DNA vaccines. Two plasmids, one expressing the N-terminal 80% E and the other expressing prM and full length E were immunogenic in intradermally inoculated mice. The vaccinated mice produced dengue-1 specific antibodies that were both neutralizing and long lasting. Data suggested that the plasmid expressing prM and full length E produced virus like particles in transfected cells, and is probably a better immunogen compared to that expressing 80% E.

Animals↗

PubMed2000

Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain.

The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides. DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR. These fragments were assembled into a full-length cDNA clone in the yeast-E. coli shuttle vector pRS424, using homologous recombination in yeast. RNA produced by in vitro transcription of this clone was infectious upon electroporation into LLCMK2 cells, as shown by cytopathic effects and detection of viral antigens by indirect immunofluorescence, and by propagation of the virus released into the culture media. Biological properties of the transcript-derived virus, such as the pattern of dengue-specific protein synthesis and growth rate in LLCMK2 or C6/36 cells, resembled those of the parent DEN1 WP virus.

Animals↗

PubMed2000

Human skin Langerhans cells are targets of dengue virus infection.

Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukocyte antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60-80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.

Blood Cells↗

PubMed2000

Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques.

A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d. -inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.

Animals↗

PubMed2000

Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus.

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.

Antibodies, Monoclonal↗

PubMed1999

Failure of secondary infection with American genotype dengue 2 to cause dengue haemorrhagic fever.

BACKGROUND: Population-based epidemiological studies have shown that infection with dengue type 2 (DEN-2) virus in individuals previously infected with a different serotype of the virus is a major risk factor for dengue haemorrhagic fever and dengue shock syndrome. However, the western hemisphere was spared epidemics of these two syndromes, until the introduction of a southeast Asian DEN-2 genotype. Possibly American DEN-2 genotype strains lacked properties necessary to cause severe disease. We report on a major epidemic of DEN-2 in Peru in 1995, about 5 years after an epidemic of DEN-1 in the same population. METHODS: In Iquitos, a city of 344,686 inhabitants in Peru, cases of dengue fever were studied prospectively from 1990. Acute phase of illness serum samples from patients were tested for virus in C6/36 cells, and virus isolates were identified by immunofluorescence. Isolates of dengue 2 virus obtained from patients during an outbreak of mild febrile illness in 1995 were sequenced to determine the genotype. Serological analysis of paired samples from the patients was done with an IgM capture ELISA and an indirect IgG ELISA. In addition, serum samples collected annually between 1993 and 1996 from a large cohort of students were tested for dengue IgG antibody by an ELISA. Serum samples from a random sample of 129 students from this cohort were tested for dengue neutralising antibodies to quantify the serotype specific infection rates. FINDINGS: Among the 129 students (aged 7-20 years in 1993) who had serum samples available before and after the epidemic, 78 (60.5%) had a secondary DEN-2 virus infection. By extrapolation, 49,266 of the 81,479 children (aged 5-14 years) in Iquitos would have experienced such infections. From previous studies, between 887 and 10,247 cases of dengue haemorrhagic fever and dengue shock syndrome would have been expected. No cases were found. DEN-2 isolates were of the American genotype. INTERPRETATION: This prospective study shows that secondary infection by the American DEN-2 genotype did not cause dengue haemorrhagic fever and dengue shock syndrome.

Adolescent↗

PubMed1999

Mayaro virus disease: an emerging mosquito-borne zoonosis in tropical South America.

This report describes the clinical, laboratory, and epidemiological findings on 27 cases of Mayaro virus (MV) disease, an emerging mosquito-borne viral illness that is endemic in rural areas of tropical South America. MV disease is a nonfatal, dengue-like illness characterized by fever, chills, headache, eye pain, generalized myalgia, arthralgia, diarrhea, vomiting, and rash of 3-5 days' duration. Severe joint pain is a prominent feature of this illness; the arthralgia sometimes persists for months and can be quite incapacitating. Cases of two visitors from the United States, who developed MV disease during visits to eastern Peru, are reported. MV disease and dengue are difficult to differentiate clinically.

Adult↗

PubMed1999

A prospective seroepidemiologic study on dengue in children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995-1996.

A prospective study on dengue (DEN) viruses was initiated in October 1995 in Gondokusuman kecamatan, Yogyakarta, Indonesia. This report presents data from the first year of the study. The studied cohort included all children 4-9 years of age living in the kecamatan. Blood samples for serology were collected from 1,837 children in October 1995 and again in October 1996. Blood samples for virus isolation and serology were collected from cohort children who were seen in municipal health clinics with febrile syndromes or admitted to hospitals with a provisional diagnosis of dengue hemorrhagic fever. Dengue serotype antibody prevalence and 1995-1996 infection rates were calculated using a single dilution (1:60) 70% plaque reduction endpoint neutralization test. Prevalence of dengue antibody at the beginning of the study was DEN 1 = 12%, DEN 2 = 16%, DEN 3 = 2%, DEN 4 = 4%, and two or more dengue infections = 22%. Total dengue antibody prevalence increased from 38% in 4-year-old children to 69% in 9-year-old children. During the observation period, primary dengue infection rates were DEN 1 = 4.8%, DEN 2 = 7.7%, DEN 3 = 4.2%, and DEN 4 = 3.4%, while two or more dengue infections occurred in 6.7% of the study population. The secondary dengue infection rate was 19.0%. From febrile cases, all four dengue viruses were isolated with DEN 3 predominating. Seven children were hospitalized, including one fatal case with a hospital diagnosis of dengue shock syndrome. Based upon presence of antibody in the initial cohort bleeding and the serologic response both weeks and several months following illness, all had secondary dengue infections. Neutralizing antibody patterns in the initial cohort bleeding and in late convalescent serum samples permitted recognition of dengue infection sequence in five patients: DEN 2-DEN 1 (3), DEN 2-DEN 4 (1), DEN 1-DEN 3 (1), and none in the sequence DEN 1-DEN 2. In the total cohort 6.5% of the observed secondary infections were of the sequence DEN 2-DEN 1, while 4.9% were DEN 1-DEN 2, a highly pathogenic sequence in previous studies. Reduced pathogenic expression of secondary DEN 2 with enhanced pathogenic expression of secondary DEN 1 infections was an unexpected finding. Further studies will be required to understand the respective contributions to pathogenicity of antibody from initial dengue infections versus the biological attributes of the second infecting dengue viruses.

Age Distribution↗

PubMed1998

Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses.

A recently described DNA vaccine for dengue (DEN) type 2 was shown to elicit high levels of neutralizing antibodies in mice. The vaccine candidate consists of the PreM and 92% of the envelope genes of DEN 2 New Guinea C strain. We further evaluated this DNA vaccine candidate by examining the effect of immuno-stimulatory CpG DNA motifs on antibody response and by studying the protective efficacy of the vaccine. The results showed that CpG motifs present in pUC 19 significantly improved the antibody response to a suboptimal dose of 3.1 micrograms of the DEN DNA vaccine. In a lethal mouse intracerebral challenge model, the vaccine provided a significant level of protection. Sixty percent of the mice immunized with the DEN DNA vaccine plus pUC 19 survived the challenge compared to only 10% in the control group that received vector plus pUC. These studies illustrate that nucleic acid immunization is a viable approach to developing a DEN vaccine and that immuno-stimulatory CpG DNA motifs can be used to lower the minimum dose required to produce an antibody response.

Adjuvants, Immunologic↗

PubMed1998

Complete nucleotide sequence analysis of a Western Pacific dengue-1 virus strain.

We have determined the complete nucleotide sequence and the deduced amino acid polypeptide sequence of the genome of a dengue-1 (DEN-1) virus strain isolated from a patient on Nauru in the Western Pacific in 1974 (West Pac 74). The complete genome is 10,735 nucleotides in length and contains a single long open reading frame of 10,176 nucleotides encoding a polyprotein of 3392 amino acids. When compared to DEN-1 Singapore S275/90, the nucleotide and amino acid sequence homology are 94% and 97.8%, respectively.

Amino Acid Sequence↗