Serologic correlates of immunity in a tetravalent reassortant rotavirus vaccine trial. US Rotavirus Vaccine Efficacy Group

The correlation of antibody responses (serum rotavirus IgA and neutralizing antibody to serotype G1-G4 human rotaviruses and rhesus rotavirus [RRV]) in a reassortant rotavirus vaccine trial with protection against rotavirus infection or disease was investigated. Most subjects administered 4 x 10(5) pfu of either the serotype G1 monovalent or serotype G1-G4 tetravalent vaccine seroconverted for at least one of the six antibodies (85% and 91%, respectively). However, fewer than one-third seroconverted to any prototype G1-G4 human rotavirus. Analyses of covariance indicated that higher prevaccination neutralizing antibody titers negatively affected postvaccination titers. Significant relationships were found between several postvaccination rotavirus antibody titers and protection, and serotype-specific correlates of protection were identified between anti-Wa titers and G1 illnesses (P = .03) and between anti-RRV titers and G3 illnesses (P < .001). Overall, however, serotype-specific immunity was no more significant than heterotypic immunity, and no specific titer of any antibody analyzed was a reliable indicator of protection.     

An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development.     

Immunological and virological analyses of persons infected by human immunodeficiency virus type 1 while participating in trials of recombinant gp120 subunit vaccines

We have studied 18 participants in phase I/II clinical trials of recombinant gp120 (rgp120) subunit vaccines (MN and SF-2) who became infected with human immunodeficiency virus type 1 (HIV-1) during the course of the trials. Of the 18 individuals, 2 had received a placebo vaccine, 9 had been immunized with MN rgp120, and seven had been immunized with SF-2 rgp120. Thirteen of the 18 infected vaccinees had received three or four immunizations prior to becoming infected. Of these, two were placebo recipients, six had received MN rgp120, and five had received SF-2 rgp120. Only 1 of the 11 rgp120 recipients who had multiple immunizations failed to develop a strong immunoglobulin G antibody response to the immunogen. However, the antibody response to rgp120 was transient, typically having a half-life of 40 to 60 days. No significant neutralizing activity against the infecting strain was detected in any of the infected individuals at any time prior to infection. Antibody titers in subjects infected despite vaccination and in noninfected subjects were not significantly different. Envelope-specific cytotoxic T-lymphocyte responses measured after infection were infrequent and weak in the nine vaccinees who were tested. HIV-1 was isolated successfully from all 18 individuals. Sixteen of these strains had a non-syncytium-inducing (NSI) phenotype, while two had a syncytium-inducing (SI) phenotype. NSI strains used the CCR5 coreceptor to enter CD4+ cells, while an SI strain from one of the vaccinees also used CXCR4. Viruses isolated from the blood of rgp120 vaccinees were indistinguishable from viruses isolated from control individuals in terms of their inherent sensitivity to neutralization by specific monoclonal antibodies and their replication rates in vitro. Furthermore, genetic sequencing of the env genes of strains infecting the vaccinees did not reveal any features that clearly distinguished these viruses from contemporary clade B viruses circulating in the United States. Thus, despite rigorous genetic analyses, using various breakdowns of the data sets, we could find no evidence that rgp120 vaccination exerted selection pressure on the infecting HIV-1 strains. The viral burdens in the infected rgp120 vaccine recipients were also determined, and they were found to be not significantly different from those in cohorts of placebo-vaccinated and nonvaccinated individuals. In summary, we conclude that vaccination with rgp120 has had,to date, no obvious beneficial or adverse effects on the individuals we have studied.     

Heterotypic protection following oral immunization with live heterologous rotaviruses in a mouse model

A Jennerian approach using live animal viruses to immunize humans is the current lead strategy for developing rotavirus vaccines. This strategy has been modified by incorporating human rotavirus VP7 genes into vaccine strains to induce serotype-specific neutralizing antibodies to human strains. However, the role of homotypic versus heterotypic immunity in protection is unclear. To investigate the importance of serotype-specific immunity in a mouse model, mice were immunized with rhesus rotavirus (RRV: G3, P5[3]), RRV-based modified Jennerian vaccine strains DxRRV (G1, P5[3]), DS1xRRV (G2, P5[3]), or ST3xRRV (G4, P5[3]), or bovine rotavirus NCDV (G6, P6[1]) and challenged with murine rotavirus ECw (G3, P[16]). Mice immunized with modified Jennerian vaccines exhibited complete to near-complete protection from challenge. NCDV-immunized mice also showed partial protection. The protection was correlated with fecal IgA levels to VP6, not serum IgG responses. Modified Jennerian vaccines induce both heterotypic and homotypic immunity in mice.     

Immune response to three doses of quadrivalent rotavirus vaccine: 1-year follow-up

Twenty-eight children who received three doses of the quadrivalent rotavirus vaccine with 4 x 10(5) plaque-forming units (p.f.u.) were followed during a year after vaccination. Serum samples were obtained and evaluated for rotavirus IgA and neutralizing antibodies against vaccine and human rotavirus strains. At the end of the study, up to 61% of the children showed an increase in circulating IgA antibody levels. Nearly all of the vaccinated children increased their neutralizing antibody titres against the vaccine strains, and 25-54% against human rotavirus serotypes. After comparing the vaccinees with a population of children naturally infected with serotype G1 in the same study area, we conclude that three doses of 4 x 10(5) p.f.u. of the quadrivalent vaccine should prepare the child against future severe rotavirus diarrhea.     

Comparative evaluation of reactogenicity and immunogenicity of two dosages of oral tetravalent rhesus rotavirus vaccine. US Rhesus Rotavirus Vaccine Study Group

OBJECTIVE: To compare the safety and immunogenicity of two dosages of tetravalent rhesus rotavirus vaccine (RRV-TV) and the effect of age at dosing. METHODS: A total of 195 infants were stratified by age into 2 groups, 6 to 12 weeks and 16 to 24 weeks, and randomly assigned to receive a single dose of placebo or RRV-TV containing either 4 x 10(5) or 4 x 10(6) plaque-forming units (pfu). Symptoms were recorded for 5 days after vaccination. Anti-rotavirus IgA and neutralizing antibody to human rotavirus serotypes G1 to G4 and RRV were measured in serum obtained pre- and postvaccination. RESULTS: Rates of fever > 38 degrees C (9%), diarrhea (6%) and vomiting (8%) were similar in all groups. IgA (69% vs. 49%, P = 0.02) and RRV (85% vs. 66%, P = 0.004) seroconversion rates were significantly higher in the 4 x 10(6) pfu vaccine group as were antibody titers to RRV (440.2 vs. 263.7, P = 0.04). Older infants demonstrated significantly higher seroconversion rates and antibody titers for IgA (71% vs. 52%, P = 0.03; and 110.6 vs. 54.8, P = 0.004) and RRV (92% vs. 66%, P = 0.05 and 498.3 vs. 205.6, P = 0.01) at either dose level than did the younger infants. There were no significant differences in seroconversion rates or antibody titers to human rotavirus types G1 to G4 between the two vaccination groups. CONCLUSIONS: RRV-TV at a dose of 4 x 10(6) pfu can be safely administered to infants 6 to 24 weeks of age. A single dose of 4 x 10(6) pfu of RRV-TV was significantly more immunogenic than a single dose of 4 x 10(5) pfu but did not improve responses to the human serotypes. Older vaccine recipients demonstrated significantly higher IgA and neutralizing antibody seroconversion rates and antibody titers than younger infants independent of dosage.     

Passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection

To determine if passively acquired antiviral antibodies modulate virus transmission and disease progression in human pediatric AIDS, the potential of pre- and postexposure passive immunization with hyperimmune serum to prevent oral simian immunodeficiency virus (SIV) infection or disease progression in newborn rhesus macaques was tested. Untreated neonates became infected after oral SIV inoculation and had high viremia, and most animals developed fatal AIDS within 3 months. In contrast, SIV hyperimmune serum given subcutaneously prior to oral SIV inoculation protected 6 newborns against infection. When this SIV hyperimmune serum was given to 3 newborns 3 weeks after oral SIV inoculation, viremia was not reduced, and all 3 infants died within 3 months of age due to AIDS and immune-complex disease. These results suggest that passively acquired antihuman immunodeficiency virus (HIV) IgG may decrease perinatal HIV transmission. However, anti-HIV IgG may not impart therapeutic benefit to infants with established HIV infection.     

Isolation of Klebsiella planticola from newborns in a neonatal ward

This is the first report on the isolation of Klebsiella planticola from neonates. Over a period of 1 year, oropharyngeal and rectal swab specimens from 131 newborns on a neonatal ward were monitored for Klebsiella spp. Thirteen strains of K. planticola could be isolated, and these represented 9% of all Klebsiella spp. found. Capsule type K27 predominated, followed by capsule type K68. Our findings suggest a significant rate of colonization by K. planticola among neonates.     

Development of a fluorescent focus identification assay using serotype-specific monoclonal antibodies for detection and quantitation of rotaviruses in a tetravalent rotavirus vaccine

A fluorescent focus identification assay (FFIDA) was developed for use in experimental studies and for quantitation of the components in a tetravalent live oral rotavirus vaccine. The assay utilizes four serotype-specific neutralizing monoclonal antibodies (MAb) to detect and quantify individual rotaviruses by immunofluorescence staining of fixed virus-infected monkey kidney cells. In mixed virus infections, all four MAb, W1 (serotype 1), 1C10 (serotype 2), R1 (serotype 3), and S4 (serotype 4), specifically stain the relevant homologous serotype without exhibiting any cross-reactivity against the other serotypes. Furthermore, the test is sensitive enough to differentiate at least twofold (0.3 log) differences in virus titer. The results of testing four individual experimental vaccine lots three or more consecutive times showed that all four lots contained similar proportions of the four vaccine strains as detected by the classical plaque neutralization identification test. The rapidity and efficiency of the FFIDA are desirable attributes that make it suitable for use in studies requiring identification and quantitation of one or more of the four major rotavirus serotypes.     

Major histocompatibility complex class I-associated vaccine protection from simian immunodeficiency virus-infected peripheral blood cells

To evaluate the effectiveness of vaccine protection from infected cells from another individual of the same species, vaccinated rhesus macaques (Macaca mulatta) were challenged with peripheral blood mononuclear cells from another animal diagnosed with acquired immune deficiency syndrome (AIDS). Half of the simian immunodeficiency virus (SIV)-vaccinated animals challenged were protected, whereas unprotected vaccinates progressed as rapidly to AIDS. Protection was unrelated to either total antibody titers to human cells, used in the production of the vaccine, to HLA antibodies or to virus neutralizing activity. However, analysis of the serotype of each animal revealed that all animals protected against cell-associated virus challenge were those which were SIV vaccinated and which shared a particular major histocompatibility complex (MHC) class I allele (Mamu-A26) with the donor of the infected cells. Cytotoxic T lymphocytes (CTL) specific for SIV envelope protein were detected in three of four protected animals vs. one of four unprotected animals, suggesting a possible role of MHC class I-restricted CTL in protection from infected blood cells. These findings have possible implications for the design of vaccines for intracellular pathogens such as human immunodeficiency virus (HIV).     

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group

OBJECTIVE: To determine the ability of live attenuated canarypox virus expressing HIV antigens to induce CD8+ cytotoxic T-cell responses and to prime for neutralizing antibody responses to boosting with purified recombinant gp120 subunit vaccine. DESIGN: A prospective, double-blind, randomized, immunogenicity and safety study was conducted in healthy adults at low risk for acquiring HIV infection and who were seronegative for HIV. METHODS: CD8+ cytotoxic T-cells directed against Env or Gag expressing target cells were measured after live recombinant canarypox-HIV-1 vaccine priming (vaccine given at days 0, 7, 14 and 21). Neutralizing antibodies were measured after subunit boosting (vaccine given at days 28 and 84). RESULTS: CD8+ CTL were induced in 64% of volunteers by the live recombinant canarypox-HIV-1 vaccine. All volunteers who received two doses of subunit vaccine after live recombinant canarypox priming developed neutralizing antibodies directed against laboratory strains of HIV-1 and seven out of eight volunteers tested developed neutralizing antibodies to the primary isolate, BZ167, but to none of eight other primary isolates. Unprimed controls had low or absent neutralizing antibodies after two doses of subunit vaccine. CONCLUSIONS: The live canarypox vector was safe, stimulated cytotoxic T-cells and primed for a vigorous neutralizing antibody response upon boosting with subunit gp120 vaccine. This vaccine combination should be evaluated further for inducing protection against HIV infection.     

Treatment of extrahepatic biliary atresia with interferon-alpha in a murine infectious model

The etiology of extrahepatic biliary atresia (EHBA) in newborns remains unknown, although a first infectious animal model with complete obstruction of the common bile duct could be established. Intraperitoneal inoculation of newborn Balb/c mice with rhesus rotavirus induced cholestasis, leading, in most cases, to biliary atresia with lethal outcome, similar to EHBA in human newborns. The influence of interferon-alpha (IFN-alpha) on the hepatotropism of rotavirus infection was investigated in this animal model. Single-dose therapy with 10000 IU of IFN-alpha protected all rhesus rotavirus-infected pups from cholestatic disease. The same dose, injected 5 d after infection, had no protective effect. Starting with onset of cholestatic symptoms, the treatment with 10000 IU of IFN-alpha daily showed good results in 29 mice. Seventy-six percent of the mice recovered after 1 wk of therapy. Histologic investigation revealed normal findings in the hepatobiliary tract of clinically normal mice. Twenty-one percent of the descendants of infected and prophylactic IFN-alpha-treated mice showed cholestatic symptoms after infection with rhesus rotavirus (79% in an untreated control group) and a milder form of the illness. In conclusion, we found that prophylactic treatment with IFN-alpha prevented the hepatobiliary system of newborn Balb/c mice from severe damage by rhesus rotavirus in this artificially designed infectious model for EHBA. Infected and icteric mice, treated for 1 wk with IFN-alpha, had good prospects for recovery and prevention of complete and irreversible occlusion of the extrahepatic bile ducts. Infected and prophylactic IFN-alpha-treated dams gave good protection to their descendants. This means that EHBA in this model could probably be averted by maternal antibodies against rotavirus.     

Fusion-competent vaccines: broad neutralization of primary isolates of HIV

Current recombinant human immunodeficiency virus (HIV) gp120 protein vaccine candidates are unable to elicit antibodies capable of neutralizing infectivity of primary isolates from patients. Here, "fusion-competent" HIV vaccine immunogens were generated that capture the transient envelope-CD4-coreceptor structures that arise during HIV binding and fusion. In a transgenic mouse immunization model, these formaldehyde-fixed whole-cell vaccines elicited antibodies capable of neutralizing infectivity of 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E. Development of these fusion-dependent immunogens may lead to a broadly effective HIV vaccine.     

Transitory neonatal hypothyroidism caused by transplacental transfer of anti-receptor antibodies of hypophyseal thyroid simulation. Case report and estimated incidence]

Transient neonatal hypothyroidism induced by transplacental transfer of thyrotropin receptor-blocking antibodies is rare, but should be diagnosed early because its course, treatment, and prognosis are different from the other forms of congenital hypothyroidism. Transient neonatal hypothyroidism should be suspected in infants with a history of maternal autoimmune thyroid disease. We describe two siblings whose mother has been treated for idiopathic primary nongoitrous hypothyroidism since adolescence. High levels of thyrotropin receptor-blocking antibodies were first detected in maternal serum at the time of the first child's birth. At the time of the second child's birth antithyroid peroxidase antibodies were found in addition to the thyrotropin receptor-blocking antibodies. Both children were clinically healthy newborns without evidence of congenital malformations. Thyroid suppression, reflected by high levels of TSH during neonatal screening, was transient in both infants. Hormonal substitution was only necessary in one child for a period of 4 months. When tested at the age of 6-7 months, maternal antibodies had completely disappeared from the infants' sera. At ages 7 and 4 years respectively the 2 children do not require treatment and show normal growth and neurodevelopmental skills. In the north-eastern part of Switzerland congenital hypothyroidism has an incidence of one in 3300 live-born infants, the most frequent form being permanent congenital hypothyroidism (1:4500). In this population, analyzed over a period of 16 years, the two cases reported represent the only observations of transient neonatal hypothyroidism due to thyrotropin receptor-blocking antibodies; the incidence can thus be estimated to be approximately 1:310,000 live newborns. In this rare condition, early recognition is pertinent in order to avoid unnecessary treatment and parental anxiety.     

Immunogenicity of group B Streptococcus type III polysaccharide-tetanus toxoid vaccine in baboons

Maternal vaccination has been proposed as a rational approach for the prevention of neonatal group B streptococcal (GBS) disease. In this study, baboons were used as a nonhuman primate model to evaluate the immunogenicity of a GBS type III glycoconjugate vaccine. Type III-specific immunoglobulin G with opsonic activity was induced after vaccination with type III polysaccharide coupled to tetanus toxoid administered with an aluminum adjuvant. This suggests that baboons could be used in evaluating maternal transfer of GBS-specific antibodies by vaccination during pregnancy.     

Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert''s disease)

Knowledge of the prevalence of congenital cytomegalovirus infection is necessary to evaluate the need for prevention. We performed a multicentre one-year study involving 11 neonatology divisions to ascertain the prevalence in Lombardy. Cytomegalovirus was isolated by culturing saliva samples from all babies born (n = 1268) of two 15-day sample periods and from 185 neonates with suspected congenital CMV based on clinical and laboratory findings and the history. The overall prevalence of congenital infection was 0.47% (6/1268) in the sample period group and 5% (9/185) in the second group. Clinical monitoring revealed sequelae in two of three children with symptomatic infection and no asymptomatic child at age two years. In a subgroup of 205 babies including 14 of the infected infants we also evaluated a test to detect cytomegalovirus DNA in the Guthrie cards obtained in neonatal screening for genetic and metabolic disorders. The test's sensitivity was 100% and specificity 98.5%, encouraging its use for early identification of infected neonates and for large epidemiological studies.     

Active cross-protection induced by a recombinant baculovirus expressing chimeric infectious bursal disease virus structural proteins

The VP2 structural gene encoded in the large genomic segment A of the variant GLS strain of infectious bursal disease virus (IBDV) was modified to encode a neutralization epitope (B69), found only on classic strains of IBDV. A chimeric cDNA clone of the large segment A, encoding VP3, VP4, and the modified variant IBDV VP2 structural proteins, was expressed in a recombinant baculovirus. The chimeric protein expressed was assessed with a panel of neutralizing monoclonal antibodies (MAbs), and it contained not only all previously MAb-defined GLS variant strain epitopes but also the B69 neutralization epitope found on classic IBDV strains. Complete active protection was afforded to specific-pathogen-free chickens by a subunit chimeric vaccine against virulent challenge with the classic IM and STC strains, as well as against the variant E/Del and GLS IBDV strains. Compared with a previously tested recombinant subunit vaccine, which incorporated unmodified baculovirus-expressed large-segment A GLS proteins, the recombinant chimeric subunit vaccine resulted in markedly improved active cross-protection against classic IBDV challenge.     

Primary influenza A virus infection induces cross-reactive antibodies that enhance uptake of virus into Fc receptor-bearing cells

Sera of young children who had had a primary infection with influenza A virus or were immunized with a live attenuated influenza A virus vaccine were examined for antibody responses that neutralized virus or enhanced uptake of virus into Fc receptor-bearing cells, because antibodies that enhance uptake of influenza virus into Fc receptor-bearing cells have been reported using mouse immune serum and monoclonal antibodies. The neutralizing antibody titers to the homologous infecting virus and to another H1N1 virus isolated several years later were higher after natural infection than after infection with the live attenuated virus. Natural infection and the attenuated vaccine induced antibodies that enhanced uptake of homologous virus and H1N1 virus isolated several years later. These results demonstrate that primary influenza A virus infection results in the induction of infection-enhancing antibodies.     

Effect of passive immunization or maternally transferred immunity on the antibody response to a genetic vaccine to rabies virus

A plasmid vector, termed pSG5rab.gp, expressing the glycoprotein of rabies virus was tested in young adult or neonatal mice in the presence of maternally transferred immunity or passively administered antibodies to rabies virus for induction of an antibody response. Mice born to rabies virus-immune dams developed an impaired antibody response to genetic immunization at 6 weeks of age, as had been previously observed upon vaccination with an inactivated viral vaccine. Similarly, mice passively immunized with hyperimmune serum showed an inhibited B-cell response upon vaccination with the pSG5rab.gp vector, resulting in both cases in vaccine failures upon challenge with a virulent strain of rabies virus. In contrast, the immune responses of mice vaccinated as neonates in the presence of maternal immunity or upon passive immunization to rabies virus with the pSG5rab.gp construct were only marginally affected.