Immunoprophylaxis of RSV infection: advancing from RSV-IGIV to palivizumab and motavizumab.

Antibodies mediate humoral immune responses and play key roles in the defense of viral infection by the recognition, neutralization, and elimination of viruses from the circulation. For the prevention of respiratory syncytial virus (RSV) infection, the natural immune response to RSV from pooled human plasma has been harvested and successfully developed as a prophylactic polyclonal RSV hyperimmune globulin, RespiGam (RSV-IGIV; MedImmune, Gaithersburg, MD). The success of RSV-IGIV validated the immunoprophylaxis approach for RSV prevention and led to the development of Synagis (palivizumab; MedImmune, Gaithersburg, MD), a humanized monoclonal antibody (mAb) that binds to the RSV F protein. Palivizumab is a potent anti-RSV mAb that is about 50-fold more potent than RSV-IGIV, and since obtaining regulatory approval in 1998 it has been used extensively to help prevent severe RSV disease in high-risk infants and children. However, a very small number of patients receiving the drug do not appear to be adequately protected. To further improve protection against RSV, we have applied a directed evolution approach to enhance the binding of palivizumab to F protein by manipulation of both the on and off rates. These efforts have yielded a more potent second-generation mAb, motavizumab, which is currently under study in phase III clinical trials. Most recently, a third generation mAb, Numax-YTE, has been generated with the intent to extend the serum half-life of the mAb in humans. If successfully developed, this drug may offer the opportunity for less frequent dosing, obviating the need for the monthly treatments that are required with palivizumab. The development of these anti-RSV approaches exemplifies the accelerated pace of drug development made possible with cutting-edge antibody engineering technologies.

Oral administration of human serum immunoglobulin in immunodeficient patients with viral gastroenteritis. A pharmacokinetic and functional analysis.

We examined the pharmacokinetics and immunological activity of human serum immunoglobulins (HSG) possessing anti-rota-virus activity which were orally administered to three children with primary immunodeficiency syndromes and prolonged gastrointestinal excretion of rotavirus. Detailed analysis of the excretion of immunoglobulins labeled with biotin or I125 revealed that approximately 50% of the recovered radioactivity was excreted in the stools over a 3-d period. Approximately half of the excreted radioactivity recovered in the stool was in a macromolecular form with immunological activity. The remainder of the recovered radioactivity was excreted in the urine as low molecular weight fragments or free iodide. In addition, immunological and chromatographic analyses revealed that the oral administration of HSG resulted in the generation of rotavirus-specific immune complexes in the gastrointestinal tract with a subsequent decrease in the presence of uncomplexed rotavirus antigen. These studies indicate that orally administered HSG can survive passage in the gastrointestinal tract in an immunologically active form, and that the oral administration of immunoglobulins with specific reactivities has potential for the prevention or treatment of gastrointestinal infections.

Experimental non-O group 1 Vibrio cholerae gastroenteritis in humans.

In this study, 27 volunteers received one of three non-O group 1 Vibrio cholerae strains in doses as high as 10(9) CFU. Only one strain (strain C) caused diarrhea: this strain was able to colonize the gastrointestinal tract, and produced a heat-stable enterotoxin (NAG-ST). Diarrhea was not seen with a strain (strain A) that colonized the intestine but did not produce NAG-ST, nor with a strain (strain B) that produced NAG-ST but did not colonize. Persons receiving strain C had diarrhea and abdominal cramps. Diarrheal stool volumes ranged from 154 to 5,397 ml; stool samples from the patient having 5,397 ml of diarrhea were tested and found to contain NAG-ST. The median incubation period for illness was 10 h. There was a suggestion that occurrence of diarrhea was dependent on inoculum size. Immune responses to homologous outer membrane proteins, lipopolysaccharide, and whole-cell lysates were demonstrable with all three strains. Our data demonstrate that V. cholerae of O groups other than 1 are able to cause severe diarrheal disease. However, not all strains are pathogenic for humans: virulence of strain C may be dependent on its ability both to colonize the intestine and to produce a toxin such as NAG-ST.

Enteric adenovirus infection and childhood diarrhea: an epidemiologic study in three clinical settings.

During a 2-year prospective study of gastroenteritis in children less than 2 years of age, the role of enteric adenovirus as a cause of infantile diarrhea was examined in three clinical settings in a case-control fashion. Using a monoclonal antibody-based enzyme-linked immunosorbent assay with specificity for adenovirus serotypes 40 and 41, enteric adenovirus was identified in 10 of 246 episodes of diarrhea in outpatients (4.1%), 13 of 211 children admitted to the hospital with diarrhea (6.2%), and 5 of 81 children in whom nosocomial diarrhea developed (6.2%), making this agent the third most commonly identified etiologic agent of diarrheal disease. Asymptomatic infections were uncommon (5 of 372 control subjects, or 1.3%) and were seen most frequently in the nosocomial setting. Cases occurred in every calendar month except March and April of each year. A syndrome of watery diarrhea of longer duration compared with other patients with diarrhea (mean 5.4 vs 3.8 days, P = .01), associated with vomiting and dehydration, was present in most cases. Compared with patients with rotavirus, patients were as likely to experience fever and dehydration and more likely to vomit. Household contact with gastroenteritis, often with a child 2 to 5 years of age, was a predisposing factor. It was concluded that enteric adenovirus is an important cause of infantile diarrhea in Baltimore children. Although far less common than rotavirus, this agent was associated with diarrheal illnesses that were at least as severe as those seen with rotavirus.

Systemic and mucosal immune responses to rhesus rotavirus vaccine MMU 18006.

Thirty-four children 3 to 20 months of age ingested either 10(5), 10(4) or 10(3) plaque-forming units of rhesus rotavirus vaccine, MMU 18006, which possesses human rotavirus serotype 3 neutralization antigen. Immune responses were evaluated by a plaque reduction neutralization (PRN) assay to rotavirus serotypes 1, 2 and 3 and by a serum IgG, IgM and IgA and fecal IgA class-specific enzyme-linked immunosorbent assay. Homotypic PRN antibody seroconversions to serotype 3 rotavirus were detected in 31 of 34 children (91%), whereas rises in heterotypic PRN antibody to human rotavirus serotypes 1 or 2 were found in only 3 of 21 (14%) (p less than 0.00000001). Thirty of the 34 vaccinated children (88%) had at least one class of rotavirus-specific serum antibody detected by enzyme-linked immunosorbent assay. A rotavirus-specific IgA coproantibody response was seen in 11 of 16 children (69%) following vaccination. Two children who had no evidence of PRN antibody to serotype 3 after vaccination had evidence of both a fecal and a serum rotavirus-specific IgA response, suggesting that in these children the response to the vaccine was primarily mucosal. These data show that orally administered rhesus rotavirus vaccine MMU 18006 elicits local intestinal immunity but produces primarily a homotypic serum neutralization response as measured by plaque reduction neutralization assays.

Diminution of the anti-polyribosylribitol phosphate response to a combined diphtheria-tetanus-acellular pertussis/Haemophilus influenzae type b vaccine by concurrent inactivated poliovirus vaccination.

BACKGROUND: Prelicensure studies of Haemophilus influenzae type b vaccines (Hib) and diphtheria-tetanus-acellular pertussis vaccines (DTaP) were evaluated with concurrent oral poliovirus vaccine (OPV). However, inactivated poliovirus vaccine (IPV) is now recommended. A trial was conducted in which infants received a DTaP and Hib vaccine, separately (+) or combined (/), with either all OPV, all IPV or sequential IPV-OPV for the primary series of vaccinations. METHODS: In this protocol 567 infants were equally randomized to receive one of the following: Reference Arm A, DTaP + Hib + OPV; Treatment Arm B, DTaP/Hib + OPV; Treatment Arm C, DTaP/Hib + IPV at 2 and 4 months and OPV at 6 months; or Treatment Arm D, DTaP/Hib + IPV. antibodies against all administered antigens were measured at 7 months of age. Children with an antibody response to Hib (anti-polyribosylribitol phosphate (anti-PRP) <0.15 microg/ml had an antibody titer repeated after the toddler booster immunization. RESULTS: A significant diminution in the anti-PRP response was observed at 7 months of age in children given two or three doses of IPV concurrently with DTaP/Hib, compared with the groups given OPV. The geometric mean concentration of anti-PRP, percentage of children with > or = 0.15 microg/ml and percentage of children with > or = 1.0 microg/ ml, respectively, were: A, 4.4, 98%, 81%; B, 3.2, 94%, 78%; C, 1.3, 86%, 58% and D, 1.2, 84%, 53%. CONCLUSION: In this trial concurrent IPV appeared to interfere with the anti-PRP response to DTaP/Hib vaccine, suggesting that introduction of new vaccines may require evaluation of immune responses to all concurrently administered vaccines.

Immunogenicity and reactogenicity of lowered doses of rhesus rotavirus vaccine strain MMU 18006 in young children.

Rhesus rotavirus oral vaccine strain MMU 18006 at a dose of 10(5) plaque-forming units (PFU), a 1:10 dilution of the original undiluted vaccine, is highly immunogenic in young children. Fevers have occurred, however, on Days 3 and 4 following vaccination. This study was conducted to determine whether febrile reactions could be eliminated and immunogenicity maintained by (1) giving smaller doses of vaccine or (2) vaccinating younger infants. Thirty-one children between 3 and 11 months of age received, in a randomized, double blind manner, either 10(4) PFU of vaccine virus, 10(3) PFU of vaccine virus or placebo. All recipients of the 10(4) PFU dose had a seroresponse; however, some degree of immunogenicity was lost with the smaller dose (10(3) PFU). Fevers were observed in recipients of both of the lowered doses of vaccine but the febrile reactions were related to the age of the vaccinee. No infant younger than 5 months of age experienced a temperature elevation, whereas the majority of children older than 5 months had fevers. Our data suggest that the lack of reaction in the younger infants correlates with the presence of prevaccination neutralizing antibody, presumably transplacentally acquired. We conclude that the rhesus rotavirus oral vaccine at a dose of 10(4) PFU is immunogenic and appears to be safe in young infants.

Palatability, reactogenicity and immunogenicity of engineered live oral cholera vaccine CVD 103-HgR in Chilean infants and toddlers.

BACKGROUND: Live oral cholera vaccine CVD 103-HgR is well-tolerated and immunogenic when administered to adults, school age children and preschool children in a single 5 x 10(9) colony-forming unit dose. Because elicitation of immune responses after administration of a single dose is exceptional for any oral vaccine in any age group, CVD 103-HgR was used as a probe to investigate the clinical acceptability, practicality and immunogenicity of this vaccine in infants and toddlers 3 to 17 months of age. METHODS: The study was undertaken successively in 12- to 17-month-olds (n = 104), 7- to 11-month-olds (n = 106) and 3- to 5-month-olds (n = 102). One-half of the subjects were randomly allocated to receive vaccine and the other one-half to receive placebo, in double blind fashion. After 2 weeks of double blind follow-up, all subjects received a dose of vaccine. Vibriocidal antibody titers were measured on coded sera collected at baseline and 2 weeks after each dosing. The buffered vaccine "cocktail" had a volume of 100 ml; subjects who ingested > or =70 ml were considered fully vaccinated. FINDINGS: Only 37% of subjects overall (25% of 3- to 5-month-olds) ingested > or =70 ml of the cocktail. The vaccine was well-tolerated with no significant differences in the rate or severity of adverse reactions after ingestion of vaccine vs. placebo. Seroconversion after ingestion of a single dose of CVD 103-HgR was similar in fully vaccinated subjects (66%) and in those who ingested a smaller fraction of the vaccine cocktail (63%). Of subjects who ingested two doses, 5 of 118 excreted vaccine organisms on Day 7 after the first dose vs. 0 of 118 after the second dose. INTERPRETATION: Single dose oral CVD 103-HgR is well-tolerated and immunogenic in infants even when a partial dose is ingested. The buffered vaccine cocktail that is readily imbibed by older children is not appealing to young infants, and improved vaccine formulations and delivery vehicles for immunizing infants must be sought.

Effect of dose and a comparison of measures of vaccine take for oral rhesus rotavirus vaccine. The Maryland Clinical Studies Group.

Ninety-six healthy infants ages 2 to 5 months received rhesus rotavirus vaccine serotype 3 (RRV) as a single dose of 10(3), 10(4) or 10(5) plaque-forming units (pfu) in this double-blinded, placebo-controlled study. Half of the infants in each dose group were also randomized to receive either 30 ml of infant formula as buffer before vaccination or were vaccinated on an empty stomach. The incidence of fever, increased stool frequency and decreased activity level was consistently higher among infants who received RRV than those who received placebo. There was no consistent increase in incidence of symptoms as the dose of RRV was increased. Possible vaccine-related side effects were increased in older vaccinees and in those with higher pre-vaccination antibody titers. The seroconversion rate and pre to postvaccination antibody rise, evaluated by enzyme-linked immunosorbent assay and by plaque reduction neutralization, correlated well. The 10(5) and 10(4) pfu RRV dose produced significantly higher rates of seroconversion and higher antibody rises than did placebo (P less than 0.001 for 10(5) and P = 0.005 for 10(4]. The 10(3) pfu dose was no more immunogenic than placebo. In the 10(4) pfu dose group 73% of infants receiving formula as a "buffer" seroconverted compared with 36% of those not receiving formula; 63% of infants partially breast-fed or formula-fed seroconverted compared with 17% of those exclusively breast-fed. These differences in seroconversion rate were largely overcome by increasing the RRV dose to 10(5) pfu. Stool (copro IgA) antibody responses were examined; of six infants showing a copro IgA response only one had seroconverted based on enzyme-linked immunosorbent assay or plaque reduction neutralization. RRV was recovered by tissue culture more frequently from the stool in those infants who received RRV 10(5) and 10(4) pfu than among those receiving 10(3) pfu or placebo (P less than 0.001).

Acute diarrhea in Baltimore children attending an outpatient clinic.

Acute diarrheal illnesses in Baltimore children younger than 2 years of age attending an outpatient clinic were studied during a 12-month period. One in five acute care visits made to the clinic by children younger than 2 years was for diarrhea, and 5% of diarrhea cases required hospitalization. With the use of comprehensive methodology, a potential etiologic agent was identified in the stool of 105 (43%) of the 246 episodes of diarrhea in cases and in 43 (28%) of the 155 controls. Viral pathogens were found in 26% of episodes, and bacterial pathogens were found in 14%. Only rotavirus, enteric adenovirus and Salmonella were significantly associated with diarrhea. Cases were more likely to have measures of socioeconomic deprivation, such as household crowding, low maternal educational level and low birth weight, when compared to controls. Racial differences in morbidity from diarrheal illnesses were observed but could be attributed to these specific sociodemographic factors. Despite the low mortality caused by infantile gastroenteritis in the United States, it remains an important public health problem. However, even with intensive investigation the etiologies remain largely unknown.

Rotavirus specific breast milk antibody in two populations and possible correlates of protection.

PIP: Since rotavirus infection is common in Caracas, Venezuela and seasonal in Rochester, New York USA, researchers analyzed at least 65 and 58 breast milk samples form women in Caracas and Rochester respectively to identify rotavirus specific antibodies. The mothers had earlier participated in Rhesus rotavirus serotype 3 (RRV) vaccine trials. The Plaque reduction neutralization assay revealed that 84.6% of breast milk samples from Venezuela had detectable antibody to RRV compared to only 50% in the US samples (p=.00009). Yet when the researchers compared US winter breast samples with the Venezuelan samples the difference was no longer significant (p=0.2). Further 50% of US nonwinter samples had rotavirus specific antibody which indicated either low level rotavirus infectious activity continuing during the nonwinter months, antigenic stimulation with cross reacting antigens, or active memory. The leading antibody responses to RRV and human P rotavirus in all breast milk samples were against VP7 antigens (62% Venezuela, 42% USA; p=.04). Only 9% of positive US samples had VP4 antigens whereas 36% of the positive Venezuelan samples had them (p=.003). 60% of the 15 4-10 month old infants in Caracas who had received RRv 10 thousand vaccine responded to the vaccine. Moreover 55% of infants who consumed breast milk with epitope blocking VP7 antibody also responded. Yet 75% of RRV vaccinated infants who took in breast milk with epitope blocking VP4 activity did not experience a seroresponse. Further 71% of those fed with breast milk negative for VP4 antibody and positive for VP7 antibody did experience a seroresponse. Thus VP4 activity in breast milk appeared to interfere with vaccine take in breast fed RRV immunized infants.^ieng

Maternal-neonatal interactions and human breast milk.

The mammary glands are a uniquely designed extension of the mucosal immune systems of the gut and bronchus. The soluble and cellular products of lactation, in which immunologic selectivity and specificity exist, link the suckling neonate irrevocably to the immunologic and infectious experience of its mother. The immune competence of the breast and products of lactation are actively and constantly in flux, dependent on the mother's hormonal, environmental, and immunologic milieu. Most of the recent human investigation has focused primarily on milk immunoglobulin specificity. More work needs to be done on the role of passively transferred cellular products, the antiviral, antiprotozoan, and antitumor capabilities of milk, and on the mechanisms by which maternal immunizations or infections may influence the outcome of host-pathogen interactions in the suckling neonate.

The immune response in primary asymptomatic and symptomatic rotavirus infection in newborn infants.

Systemic and mucosal immune responses were determined using an enzyme-linked immunosorbent assay in 18 infants (7-86 days old) experiencing primary rotavirus infections over two winters. Fifteen infected infants were asymptomatic; 3 had diarrhea. Neutralization antibody (NA) was assayed in two asymptomatic infants who had a serotype 1 isolate identified. Seven asymptomatic infants had sera available for analysis; none had IgG, 1 had IgM, but 4 had IgA antibody responses. Neither tested infant had a serotype 1 NA rise. In the 3 symptomatic infants, 1 had IgG, 2 had IgM, and all had IgA serum antibody responses detected. Eleven (73%) of 15 asymptomatic and all symptomatic infants had a rotavirus-specific fecal antibody response. These findings identify IgA as an important antibody in primary rotavirus infection in very young infants. The predominance of this antibody in asymptomatic infants suggests that their responses (and protection on subsequent reexposure) may be primarily mucosal.

Asymptomatic infections due to wild-type rotavirus may prime for a heterotypic response to vaccination with rhesus rotavirus.

Variable homotypic and heterotypic protection has been observed following oral vaccination with rhesus rotavirus vaccine. Natural asymptomatic infections may enhance the efficacy of rhesus rotavirus vaccine. Asymptomatic seroconversion before and during the epidemic rotavirus season in Rochester, New York, was examined in a trial of 190 2- to 5-month-old infants receiving rotavirus and placebo vaccines. Six (37.5%) of 16 placebo recipients seroconverted; four of the six did so 2 to 8 months before the Rochester rotavirus season. Among the recipients of rotavirus vaccine, eight seroconverted before the rotavirus season as a consequence of asymptomatic infection. Four of the eight had not seroconverted after rotavirus vaccination, and four seroconverted from asymptomatic infection as well as from vaccination. Twelve rotavirus vaccinees had an asymptomatic seroconversion concurrent with the epidemic rotavirus season in Rochester: eight seroconverted following apparent vaccine failure, and four seroconverted following an apparent vaccine take. Six vaccinated infants with asymptomatic seroconversion during the rotavirus season had significant rises in the level of IgA antibody determined by ELISA, thereby suggesting preferential stimulation of memory cells of IgA. The possibility that variable heterotypic protection following oral rotavirus vaccination may be due to enhanced immunogenicity as a consequence of prior or concurrent infection with circulating wild-type rotaviruses appears to be supported by the findings of this report.

Molecular epidemiology of rotavirus infection in a room for convalescing newborns.

In a prospective study, all patients admitted to a unit for convalescing newborns on an infant-toddler ward between January and April 1985 were tested every other day for rotavirus (RV) excretion by using an enzyme-linked immunosorbent assay. RNA electrophoresis of the RV strains identified in this population were analyzed to determine whether specific strains of virus were associated with infections in newborns. RV strains infecting older, symptomatic children from the infant-toddler ward were used for comparison. Among the 39 patients enrolled in the study, RV was detected in the stools of 13 (33%), 11 (86%) of whom were asymptomatic. Three different electropherotypes were seen among the newborns who were RV positive; these electropherotypes were identical to those identified in older children with community-acquired diarrhea and nosocomial-acquired diarrhea who were hospitalized on the infant-toddler ward during the same study period. Each of the three strains was first identified in the general ward and subsequently detected in the room with newborns within three to six days. We failed to identify a particular strain that exclusively infected newborns.

Immunologic response to oral cholera vaccination in a crossover study: a novel placebo effect.

The authors conducted a two-period crossover study of the reactogenicity and immunogenicity of live oral cholera vaccine CVD 103-HgR among US university students. Subjects ingested 5 x 10(8) colony forming units of either killed Escherichia coli K12 placebo or vaccine, followed by the opposite treatment one week later. Surprisingly, the dynamics of the immunologic response were influenced by prior ingestion of placebo. Subjects who received placebo first showed stronger vibriocidal antibody responses 2 weeks after vaccination compared with subjects who received vaccine first; this same pattern was seen for antitoxin titers. The authors suggest that ingestion of E. coli K12 one week prior to immunization boosts the immunologic response to vaccine by an unknown mechanism. Future crossover studies that examine immunologic outcomes might be designed to explore the ubiquity of such an effect.

Serum antibody responses of divers to waterborne pathogens.

To assess the significance of exposure of divers to waterborne pathogens, specific immunoglobulin G serum antibody responses to Pseudomonas and Aeromonas isolates recovered from dive sites from the respiratory tracts of nine experienced divers and seven diving trainees working in the Chesapeake Bay area over a 6- to 18-month period were measured. A significant increase in the frequency of isolation of these organisms from respiratory surfaces both groups of divers after each dive was noted, with the divers' ears being the predominant recovery site (48%; P < 10(-8), chi-square). The acute serum responses of the majority of experienced divers (83%) showed evidence of preexisting antibody to these potential pathogens, whereas the acute serum response of only 32% of naive divers showed such evidence (P < 10(-8), chi-square). Six months into their training, the rate of seroresponse of the trainees to organisms recovered after their first dives increased to 61% (P = 0.003, chi-square), suggesting that repeated exposure in necessary for generation of a specific systemic immunologic response. The rate of acquisition of a new seroresponse to recovered organisms was approximately 12% per dive for both groups of divers, suggesting that there is continuous exposure to, and infection with, new strains present in the water during dives. These data suggest that, in cases in which systemic antibody is important for protection, there are various levels of susceptibility to waterborne potential pathogens in both experienced and inexperienced divers.

Experimental human infections with Giardia lamblia.

Fifteen healthy volunteers were inoculated enterally with trophozoites of two distinct human isolates of Giardia lamblia, GS/M and Isr. Each of two groups of five volunteers were inoculated with 50,000 (GS/M or Isr) trophozoites. All of the volunteers inoculated with GS/M and none of the volunteers inoculated with Isr became infected. Three of five volunteers infected with GS/M became ill, including two who had diarrhea and typical symptoms of giardiasis. In the second study, three patients who were previously infected with GS/M and treated were rechallenged 12 weeks after the first inoculation, together with five new control volunteers. All of the latter group became infected, and two developed loose stools; two rechallenged volunteers became reinfected but were asymptomatic, and a third was retrospectively found to be infected at the time of challenge. Serum IgM, IgG, and IgA antibody responses to Giardia and intestinal fluid IgA antibody responses to Giardia occurred in 100%, 70%, 60%, and 50%, respectively, of infected volunteers. These studies fulfill Koch's postulates and demonstrate strain variation in the pathogenicity of Giardia infections in humans.

Effect of immunization against rubella on lactation products. I. Development and characterization of specific immunologic reactivity in breast milk.

The development of an immune response to rubella virus in milk, serum, and nasopharyngeal secretions was studied in lactating postpartum women after immunization with HPV-77 De5 or RA 27/3 live, attenuated rubella virus vaccine administered subcutaneously or intranasally. Over 69% of the women shed virus in milk after immunization. A predictable nasopharyngeal IgA and serum IgG antibody response to rubella virus was observed after subcutaneous or intranasal immunization with RA 27/3 vaccine. Little or no nasopharyngeal antibody response was seen after subcutaneous immunization with HPV-77 DE5 vaccine. A virus-specific IgA antibody response in milk was seen in all women. The presence of rubella virus in breast milk seemed to potentiate the peak levels of virus-specific antibody in the milk. Cellular immune reactivity to rubella virus in milk was observed in all vaccine groups. Thus, the virus-specific immune response induced in human milk after immunization with rubella virus vaccine may be intimately linked with the reactivity in bronchial lymphoid tissue.