Emmanuel B. Walter

High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V(H) and V(L)) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V(H) and V(L) genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V(H) or V(L) genes. The utility of these Ig gene expression cassettes was established using synthetic V(H) or V(L) genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V(H) and V(L) genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

Human Responses to Influenza Vaccination Show Seroconversion Signatures and Convergent Antibody Rearrangements

B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individuals secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

Background During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. Methods and Findings To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. Conclusion The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

HIV-1 sensitivity to zidovudine and clinical outcome in children

In adults with the acquired immunodeficiency syndrome, long-term monotherapy with zidovudine selects for human immunodeficiency virus type 1 (HIV-1) strains with substantially reduced in-vitro susceptibility to the drug. We have assessed the relation between in-vitro resistance to zidovudine and clinical outcome in children, in whom disease progression is more rapid than in adults. We studied 23 children with symptoms of HIV-1 disease during extended monotherapy with zidovudine. An in-vitro assay was used to determine the concentration of zidovudine required to inhibit by 50% the replication of viral isolates (IC50) obtained after 9 to 39 months of treatment. Viral stocks of high enough titre to yield reproducible results were obtained from 19 of the children. During the following 6 months of therapy, 9 children were stable, 7 deteriorated, and 3 died. There was a highly significant relation between decreased zidovudine susceptibility and poor clinical outcome (p less than 0.001) but no relation between IC50 and age at start of therapy or length of time on treatment. Age-adjusted CD4 lymphocyte counts were lower at the start of treatment (p = 0.02) and at the time of sampling (p = 0.01) in children whose viral isolates had an increased zidovudine IC50. Initial serum p24 antigen levels were not predictive of subsequent emergence of resistant virus, but at the time of sampling for viral sensitivity higher p24 antigen levels were associated with raised IC50 (p = 0.004). The findings suggest that most children who become unresponsive to monotherapy with zidovudine, as judged by clinical criteria, will have changes in in-vitro sensitivity to the drug. In these children, an alternative antiretroviral therapy should be considered.

Postlicensure study of varicella vaccine effectiveness in a day-care setting.

BACKGROUND Varicella vaccine has been licensed for use in the United States since the spring of 1995. The acceptance of the vaccine and its effect on varicella incidence in children is important. AIM To document the effectiveness of the varicella vaccine in children attending day care in 11 centers in North Carolina. METHODS A dynamic cohort study design was used in 11 day-care centers in North Carolina. Multiple cross-sectional evaluations were performed and children were noted to be vaccinated or not and diseased or not. Vaccine effectiveness was estimated by comparing the varicella attack rate in the vaccinated with the varicella attack rate in the unvaccinated. Person time was used as the denominator for all calculations. RESULTS During the study period February 1, 1996, to September 1, 1997, 134 cases of varicella occurred in the unvaccinated and 11 cases occurred in the vaccinated children. The attack rates in the vaccinated and unvaccinated were 2.49 and 14.66, respectively, for an overall vaccine effectiveness of 83% for mild/moderate disease. CONCLUSIONS In the day-care setting varicella vaccine demonstrated benefit in preventing and modifying wild-type varicella disease.

A comparison of 2 influenza vaccine schedules in 6- to 23-month-old children.

Background. Trivalent inactivated influenza vaccine (TIV) is recommended for all children ages 6 to 23 months. Delivering 2 doses of TIV at least 4 weeks apart to young children receiving this vaccine for the first time is challenging. Methods. We compared the immunogenicity and reactogenicity of the standard 2-dose regimen of TIV administered in the fall with an early schedule of a single spring dose followed by a fall dose of the same vaccine in healthy toddlers 6 to 23 months of age. Children were recruited in the spring to be randomized into either the standard or early schedule. An additional group was also enrolled in the fall as part of a nonrandomized standard comparison group. The 2002–2003 licensed TIV was administered in the spring; the fall 2003–2004 vaccine contained the same 3 antigenic components. Reactogenicity was assessed by parental diaries and telephone surveillance. Blood was obtained after the second dose of TIV for all children. The primary outcome measure was antibody response to influenza A/H1N1, A/H3N2, and B after 2 doses of vaccine, as determined by hemagglutination-inhibition titers ≥1:32 and geometric mean titer (GMT). Results. Two hundred nineteen children were randomized to receive either the standard or early TIV schedule; 40 additional children were enrolled in the fall in the nonrandomized standard group. Response rates in the combined standard versus early groups were similar overall: 78% (GMT: 48) vs 76% (GMT: 57) to H1N1, 89% (GMT: 115) vs 88% (GMT: 129) to H3N2, and 52% (GMT: 24) vs 60% (GMT: 28) to B. Reactogenicity after TIV in both groups of children was minimal and did not differ by dose, age, or time between doses. Reaction rates were higher in those receiving TIV and concomitant vaccines compared with those receiving TIV alone. Overall rates of fever >38°C axillary and injection-site pain, redness, or swelling were 5.4%, 3.1%, 0.9%, and 1.1%, respectively. Conclusions. When the spring and fall influenza vaccines had the same 3 antigenic components, the early vaccine schedule resulted in similar immunogenicity and reactogenicity compared with the standard schedule. When the vaccine components do not change between years, initiating influenza vaccine in the spring at the time of routine office visits would facilitate full immunization of children against influenza earlier in the season.

Safety and Immunogenicity of an Inactivated Influenza A/H5N1 Vaccine Given with or without Aluminum Hydroxide to Healthy Adults: Results of a Phase I–II Randomized Clinical Trial

BACKGROUND Dose-sparing strategies are being explored for vaccines against pandemic influenza. We evaluated the dose-sparing potential of aluminum hydroxide (AlOH) adjuvant. METHODS A total of 600 healthy subjects (age, 18-49 years) were randomized to receive 2 vaccinations 1 month apart with subvirion inactivated influenza A/H5N1 vaccine containing 7.5, 15, or 45 microg of hemagglutinin (HA), with or without 600 microg of aluminum hydroxide (AlOH), or 3.75 microg of HA, with or without 300 microg of AlOH. Serum specimens were obtained for antibody assays before and 1 month after each vaccination. RESULTS All formulations were safe. Injection site discomfort was more frequent in groups given vaccines with AlOH. Dose-related increases in antibody responses were noted after both vaccinations (P< .001) geometric mean titers of hemagglutination inhibition antibody in vaccines with and without AlOH, respectively, were 5.4 and 5.4 for subjects who received 3.75 microg of HA, 7.7 and 5.3 for those who received 7.5 microg of HA, 8.1 and 8.5 for those who received 15 microg of HA, and 14.8 and 12 for those who received 45 microg of HA. A > or =4-fold increase in titer was observed in 2% and 2% of subjects who received 3.75 microg of HA with or without AlOH, respectively; in 14% and 0% who received 7 microg of HA; in 14% and 13% who received 15 microg of HA; and in 33% and 25% who received 45 microg of HA. Addition of AlOH enhanced responses only for subjects who received 7.5 microg of HA, but responses in subjects who received 7.5 microg of HA without AlOH were unexpectedly low. CONCLUSION Overall, a meaningful beneficial effect of AlOH adjuvant was not observed. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00296634 .

Psychosocial and Behavioral Correlates of Depression Among HIV-Infected Pregnant Women

This study addressed two aims: (1) to assess the level of depressive symptoms among pregnant, HIV-infected racial and ethnic minority women and (2) to identify potentially modifiable factors associated with prenatal depression in order to foster proactive clinical screening and intervention for these women. Baseline interview data collected from HIV-infected women participating in the Perinatal Guidelines Evaluation Project were analyzed. Participants were from prenatal clinics in four areas representative of the U. S. HIV/AIDS epidemic among women. Of the final sample (n = 307), 280 were minorities (218 blacks [African American and Caribbean], 62 Hispanic). Standardized interviews assessed potential psychosocial factors associated with pregnancy-related depression and psychological distress (life stressors, inadequate social support, and ineffective coping skills) in a population for whom little work has been done. Depressive symptomatology was considerable, despite excluding somatic items in order to avoid confounding from prenatal or HIV-related physical symptoms. The psychosocial factors significantly predicted the level of prenatal depressive symptoms beyond the effects of demographic and health-related factors. Perceived stress, social isolation, and disengagement coping were associated with greater depression, positive partner support with lower depression. These findings demonstrate that psychosocial and behavioral factors amenable to clinical intervention are associated with prenatal depression among women of color with HIV. Routine screening to identify those currently depressed or at risk for depression should be integrated into prenatal HIV-care settings to target issues most needing intervention.

Influenza Vaccine Immunogenicity in 6- to 23-Month-Old Children: Are Identical Antigens Necessary for Priming?

OBJECTIVES. Immunoprophylaxis with influenza vaccine is the primary method for reducing the effect of influenza on children, and inactivated influenza vaccine has been shown to be safe and effective in children. The Advisory Committee on Immunization Practices recommends that children 6 to 23 months of age who are receiving trivalent inactivated influenza vaccine for the first time be given 2 doses; however, delivering 2 doses of trivalent inactivated influenza vaccine ≥4 weeks apart each fall can be logistically challenging. We evaluated an alternate spring dosing schedule to assess whether a spring dose of trivalent inactivated influenza vaccine was capable of “priming” the immune response to a fall dose of trivalent inactivated influenza vaccine containing 2 different antigens. PATIENTS AND METHODS. Healthy children born between November 1, 2002, and December 31, 2003, were recruited in the spring and randomly assigned to either the alternate spring schedule or standard fall schedule. The 2003–2004 licensed trivalent inactivated influenza vaccine was administered in the spring; the fall 2004–2005 vaccine had the same A/H1N1 antigen but contained drifted A/H3N2 antigen and B antigen with a major change in strain lineage. Reactogenicity was assessed by parental diaries and telephone surveillance. Blood was obtained after the second dose of trivalent inactivated influenza vaccine for all of the children and after the first dose of trivalent inactivated influenza vaccine in the fall group. The primary outcome of this study was to demonstrate noninferiority of the antibody response after a spring-fall dosing schedule compared with the standard fall dosing schedule. Noninferiority was based on the proportion of subjects in each group achieving a hemagglutination-inhibition antibody titer of ≥1:32 after vaccination to 2 of the 3 antigens (H1N2, H3N2, and B) contained in the 2004–2005 vaccine. For each antigen, the antibody response was proposed to be noninferior if, within the upper bound of 95% confidence interval, there was <15% difference between the proportion of children in the fall and spring groups with postvaccination titers ≥1:32. RESULTS. A total of 468 children were randomly assigned to either the spring (n = 233) or fall (n = 235) trivalent inactivated influenza vaccine schedule. Excellent response rates to A/H1N1, as measured by antibody levels ≥1:32, were noted in both the spring (86%) and fall groups (93%). The A/H1N1 response rate of the spring group was noninferior to that of the fall group. Noninferiority of the spring schedule was not met with respect to the other 2 influenza antigens: for A/H3N2 the response was 70% in the spring group versus 83% for the fall group, and the response to B was 39% in the spring group versus 88% for the fall group. After 2 doses of vaccine, the geometric mean antibody titers also were less robust in the spring group for both A/H3N2 and B antigens. For each of the 3 vaccine antigens, the respective geometric mean antibody titers for the spring group versus the fall group were: A/H1N1, 79.5 ± 3.3 and 91.9 ± 2.6; A/H3N2, 57.1 ± 4.1 and 77.8 ± 3.7; and B, 18.0 ± 2.4 and 61.6 ± 2.5. However, a significantly higher proportion of children in the spring group achieved potentially protective levels of antibody to all 3 antigens after their first fall dose of trivalent inactivated influenza vaccine than children in the fall group after receiving their first fall dose. For influenza A/H1N1, there was an antibody level ≥1:32 in 86% of children in the spring group versus 55% of children in the fall group. Likewise, for influenza A/H3N2, 70% of children in the spring group and 47% of children in the fall group had antibody levels >1:32; for influenza B, the proportions were 39% of children in the spring group and 16% of children in the fall group. Reactogenicity after trivalent inactivated influenza vaccine in both groups of children was minimal and did not differ by dose. CONCLUSIONS. Although the immune response to the identical A/H1N1 vaccine antigen was similar in both groups, priming with different A/H3N2 antigens and B antigens in the spring produced a lower immune response to both antigens than that shown in children who received 2 doses of the same vaccine in the fall. However, ∼70% of children in the spring group had a protective response to the H3N2 antigen after 2 doses. Initiating influenza immunization in the spring was superior to 1 dose of trivalent inactivated influenza vaccine in the fall. The goal of delivering 2 doses of influenza vaccine a month apart to vaccine-naive children within the narrow flu vaccination season is a challenge not yet met; thus far, only about half of children aged 6 to 23 months of age are receiving influenza vaccine. By using the spring schedule, we were able to administer 2 doses of trivalent inactivated influenza vaccine to a higher proportion of children earlier in the influenza vaccination season. In years when there is an ample supply of trivalent inactivated influenza vaccine, and vaccine remains at the end of the season, priming influenza vaccine-naive infants with a spring dose will lead to the earlier protection of a higher proportion of infants in the fall. This strategy may be particularly advantageous when there is an early start to an influenza season as occurred in the fall of 2003. A priming dose of influenza vaccine in the spring may also offer other advantages. Many vaccine-naive children may miss the second dose of fall trivalent inactivated influenza vaccine because of vaccine shortages or for other reasons, such as the potential implementation of new antigens at a late date. Even with seasonal changes in influenza vaccine antigens, by giving a springtime dose of trivalent inactivated influenza vaccine, such children would be more protected against influenza than would children who were only able to receive 1 dose in the fall. In summary, our data suggest that identical influenza antigens are not necessary for priming vaccine-naive children and that innovative uses of influenza vaccine, such as a springtime dose of vaccine, could assist in earlier and more complete immunization of young children.

Over five-year follow-up of Oka/Merck varicella vaccine recipients in 465 infants and adolescents.

A total of 465 healthy infants and adolescents ages 12 months to 17 years without a known history of varicella or recent exposure to varicella-zoster virus VZV were immunized with live attenuated Oka/Merck varicella vaccine from November, 1984, through April, 1989. The vaccine administered was from 1 of 7 production lots containing from 950 to 3265 plaque-forming units and was well-tolerated with few side effects. The seroconversion rate for seronegative subjects was 94.6% (403 of 426). This varied by lot from 85% (950 plaque-forming units) to 100% (3010 and 3265 plaque-forming units). Breakthrough disease after exposure to varicella in seroconverters during 5 to 10 years of follow-up was 18.6% (75 of 403). The breakthrough disease was characterized by a maculopapular rash with a median of 35 lesions, most of which were macules. Breakthrough disease lasted a median of 5 days and the median temperature was 99°F; 65.3% (49 of 75) of subjects were afebrile and 2.7% (2 of 75) of subjects had temperatures of > 102.9°F. Varicella vaccine provides excellent (94.6%) seroconversion, and most children who developed breakthrough disease (18.6%) experienced a modified, milder form of illness than has been observed with natural varicella in unvaccinated subjects.