Detection of Anti-SARS-CoV-2-S2 IgG Is More Sensitive Than Anti-RBD IgG in Identifying Asymptomatic COVID-19 Patients.

Characterizing the serologic features of asymptomatic SARS-CoV-2 infection is imperative to improve diagnostics and control of SARS-CoV-2 transmission. In this study, we evaluated the antibody profiles in 272 plasma samples collected from 59 COVID-19 patients, consisting of 18 asymptomatic patients, 33 mildly ill patients and 8 severely ill patients. We measured the IgG against five viral structural proteins, different isotypes of immunoglobulins against the Receptor Binding Domain (RBD) protein, and neutralizing antibodies. The results showed that the overall antibody response was lower in asymptomatic infections than in symptomatic infections throughout the disease course. In contrast to symptomatic patients, asymptomatic patients showed a dominant IgG-response towards the RBD protein, but not IgM and IgA. Neutralizing antibody titers had linear correlations with IgA/IgM/IgG levels against SARS-CoV-2-RBD, as well as with IgG levels against multiple SARS-CoV-2 structural proteins, especially with anti-RBD or anti-S2 IgG. In addition, the sensitivity of anti-S2-IgG is better in identifying asymptomatic infections at early time post infection compared to anti-RBD-IgG. These data suggest that asymptomatic infections elicit weaker antibody responses, and primarily induce IgG antibody responses rather than IgA or IgM antibody responses. Detection of IgG against the S2 protein could supplement nucleic acid testing to identify asymptomatic patients. This study provides an antibody detection scheme for asymptomatic infections, which may contribute to epidemic prevention and control.

Delayed neutralizing antibody response in the acute phase correlates with severe progression of COVID-19.

Adaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dynamics remain largely unknown. The neutralizing antibody (NAb) levels in patients with coronavirus disease 2019 (COVID-19) are helpful for understanding the pathology. Using SARS-CoV-2 pseudotyped virus, serum sample neutralization values in symptomatic COVID-19 patients were measured using the chemiluminescence reduction neutralization test (CRNT). At least two sequential serum samples collected during hospitalization were analyzed to assess NAbs neutralizing activity dynamics at different time points. Of the 11 patients, four (36.4%), six (54.5%), and one (9.1%) had moderate, severe, and critical disease, respectively. Fifty percent neutralization (N50%-CRNT) was observed upon admission in 90.9% (10/11); all patients acquired neutralizing activity 2-12 days after onset. In patients with moderate disease, neutralization was observed at earliest within two days after symptom onset. In patients with severe-to-critical disease, neutralization activity increased, plateauing 9-16 days after onset. Neutralization activity on admission was significantly higher in patients with moderate disease than in patients with severe-to-critical disease (relative % of infectivity, 6.4% vs. 41.1%; P = .011). Neutralization activity on admission inversely correlated with disease severity. The rapid NAb response may play a crucial role in preventing the progression of COVID-19.

Previous SARS-CoV-2 Infection Increases B.1.1.7 Cross-Neutralization by Vaccinated Individuals.

With the spread of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a need to assess the protection conferred by both previous infections and current vaccination. Here we tested the neutralizing activity of infected and/or vaccinated individuals against pseudoviruses expressing the spike of the original SARS-CoV-2 isolate Wuhan-Hu-1 (WH1), the D614G mutant and the B.1.1.7 variant. Our data show that parameters of natural infection (time from infection and nature of the infecting variant) determined cross-neutralization. Uninfected vaccinees showed a small reduction in neutralization against the B.1.1.7 variant compared to both the WH1 strain and the D614G mutant. Interestingly, upon vaccination, previously infected individuals developed more robust neutralizing responses against B.1.1.7, suggesting that vaccines can boost the neutralization breadth conferred by natural infection.

COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants.

As SARS-CoV-2 has been circulating for over a year, dozens of vaccine candidates are under development or in clinical use. The BNT162b2 mRNA COVID-19 vaccine induces spike protein-specific neutralizing antibodies associated with protective immunity. The emergence of the B.1.1.7 and B.1.351 variants has raised concerns of reduced vaccine efficacy and increased re-infection rates. Here we show, that after the second dose, the sera of BNT162b2-vaccinated health care workers (n = 180) effectively neutralize the SARS-CoV-2 variant with the D614G substitution and the B.1.1.7 variant, whereas the neutralization of the B.1.351 variant is five-fold reduced. Despite the reduction, 92% of the seronegative vaccinees have a neutralization titre of >20 for the B.1.351 variant indicating some protection. The vaccinees' neutralization titres exceeded those of recovered non-hospitalized COVID-19 patients. Our work provides evidence that the second dose of the BNT162b2 vaccine induces cross-neutralization of at least some of the circulating SARS-CoV-2 variants.

Age-Dependent Evaluation of Immunoglobulin G Response after Chikungunya Virus Infection.

Current chikungunya antibody prevalence and titers are likely to differ based on the exposure rates before the 2006 reemergence in India. For vaccine usage, such data are of immense importance. This study addresses age-stratified IgG titers and its subtypes in Pune, India, endemic for the disease. 170 age-stratified serum pools from 791 individuals with prior chikungunya exposure, and 15 samples from acute disease phase were analyzed. An indirect ELISA based on inactivated chikungunya virus was used to determine anti-CHIKV-IgG and its subtypes. Neutralizing antibody titers (plaque reduction neutralization test [PRNT]) were compared with binding antibody titers (ELISA). Anti-CHIKV-IgG titers along with IgG1 and IgG4 increased till the age-group of until 11-15 years and remained comparable thereafter till > 65 years. IgG1 was the predominant IgG subtype detected in all the pools, whereas IgG4 was present in 151/170 pools. Strong positive correlation of IgG1 was obtained with CHIKV-PRNT50 titers. None of the sample had anti-CHIKV-IgG2, whereas five pools had IgG3 antibody. In the acute-phase serum sample, IgG1 was present in all the samples, whereas IgG4 was present in 8/15 samples. IgG4 was predominant in four samples. During acute phase and at different times postinfection, IgG1 circulated in high titers followed by IgG4. Higher antibody titers in adults reflect reexposures. The data will prove useful in assessing immune response to CHIKV vaccine in relation to IgG subtype.

Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients.

The association of mortality with the early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improve sensitivity and minimize interference. Disposable cartridges containing pre-dispensed reagents require no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher TAb and SNAb positivity rates and more robust antibody responses at patient's initial hospital presentation were seen in inpatients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who had negative TAb and/or SNAb at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow the detection of early SARS-CoV-2 antibodies which associate with mortality.

Correlation of SARS-CoV-2 Neutralizing Antibodies to an Automated Chemiluminescent Serological Immunoassay.

INTRODUCTION: Neutralizing antibodies (NAbs) are capable of binding to a virus to render it incapable of infection. The ability of commercially available SARS-CoV-2 serological tests to detect NAbs has not been widely reported. We sought to correlate the antibodies detected by an automated chemiluminescent immunoassay with NAbs. METHODS: Residual serum samples from 35 patients that had a positive antibody test using the LIAISON® SARS-CoV-2 S1/S2 IgG chemiluminescent immunoassay and 2 antibody-negative control sera were tested for NAbs using a plaque reduction neutralization test (PRNT). RESULTS: NAbs were detected in 66% (23/35) of the antibody-positive samples. The immunoassay signal value ranged from 21.7 to 131.3 AU/mL (median, 90.5) with significant correlation between it and the PRNT (r = 0.61, P = 0.002). In the samples without NAbs, the immunoassay signal ranged from 16.3 to 66.2 AU/mL (median, 27.2). An immunoassay signal cutoff of >41 AU/mL was 91% sensitive and 92% specific for the detection of NAbs. DISCUSSION: It is important that correlates of immunity to SARS-CoV-2 be identified and NAbs are considered to be central indicators of such. PRNT is the gold-standard test for identifying NAbs but it cannot be used for large-scale testing of populations. It is necessary to establish relationships between it and widely used commercial serological assays for SARS-CoV-2.

Accuracy and efficacy of pre-dengue vaccination screening for previous dengue infection with five commercially available immunoassays: a retrospective analysis of phase 3 efficacy trials.

BACKGROUND: The tetravalent dengue vaccine (CYD-TDV) has been shown to provide protection against dengue disease over 5-year follow-up in participants with previous dengue infection, but increased the risk of dengue hospitalisation and severe dengue during long-term follow-up in those without previous dengue infection. WHO recommended pre-vaccination screening to identify those with previous dengue infection (ie, dengue seropositive) who would benefit from vaccination. We re-evaluated CYD-TDV efficacy in those identified as dengue seropositive using five commercially available immunoassays, and assessed immunoassay performance. METHODS: We included participants in the immunogenicity subsets of the phase 3 CYD14 (NCT01373281) and CYD15 (NCT01374516) CYD-TDV efficacy trials, which enrolled children aged 2-16 years in 2011-12 in five countries in the Asia-Pacific region (CYD14) and five Latin American countries (CYD15). Participants assessed had received at least one injection of study drug (CYD-TDV or placebo) and had baseline samples available. We tested baseline samples by IgG-based immunoassays to classify baseline dengue serostatus, using two ELISAs (EUROIMMUN and Panbio) and three rapid diagnostic tests (RDTs; TELL ME FAST, SD BIOLINE, and OnSite). Vaccine efficacy in preventing symptomatic, hospitalised, and severe virologically confirmed dengue was determined for participants who tested positive by each immunoassay. The specificity and sensitivity of each immunoassay was determined as percentage negative and positive agreement compared with the reference algorithm, which used dengue plaque reduction neutralisation test with 50% and 90% cutoffs and non-structural protein 1 IgG ELISA results to assign baseline serostatus. FINDINGS: Samples were available for 3967 participants, 2735 (69·0%) of whom were classified as seropositive by the reference algorithm. Vaccine efficacy against symptomatic virologically confirmed dengue in immunoassay-positive participants was high across all five immunoassays (EUROIMMUN ELISA 88·2% [95% CI 77·3 to 93·9], Panbio ELISA 87·6% [76·7 to 93·4], TELL ME FAST RDT 88·8% [67·0 to 96·2], SD BIOLINE RDT 82·8% [66·9 to 91·1], and OnSite RDT 89·7% [64·6 to 97·0]), as was vaccine efficacy against hospitalised virologically confirmed dengue (EUROIMMUN-ELISA 72·8% [38·9 to 87·9], Panbio ELISA 77·5% [52·8 to 89·3], TELL ME FAST RDT 92·4% [37·8 to 99·1], SD BIOLINE RDT 87·2% [54·5 to 96·4], and OnSite RDT 73·7% [-5·1 to 93·4]) and severe virologically confirmed dengue (EUROIMMUN ELISA 86·9% [-16·8 to 98·5], Panbio ELISA 91·3% [27·6 to 99·0], TELL ME FAST RDT 100·0% [not estimable to 100·0%], SD BIOLINE RDT 89·4% [9·6 to 98·8], and OnSite RDT 73·4% [-193·7 to 97·6]). The immunoassays exhibited high specificity (≥98·8% for all immunoassays apart from SD BIOLINE RDT) but variable sensitivities, with higher sensitivities observed for the ELISAs (EUROIMMUN 89·2% [87·9 to 90·3] and Panbio 92·5 [91·4 to 93·5]) than the RDTs (TELL ME FAST 52·5% [50·6 to 54·4], SD BIOLINE 71·1% [69·3 to 72·8], and OnSite 47·6% [45·7 to 49·5]). INTERPRETATION: Our findings suggest that these immunoassays could be used for pre-vaccination screening for CYD-TDV as tools to assist risk stratification until more sensitive and convenient tests become available. FUNDING: Sanofi Pasteur.

Neutralizing antibody rather than cellular immune response is maintained for nearly 20 years among Japanese encephalitis SA14-14-2 vaccinees in an endemic setting.

Japanese encephalitis (JE), caused by infection with Japanese encephalitis virus (JEV), is the most important viral encephalitis in Asia. JE incidence has significantly decreased by immunization with live-attenuated vaccine SA14-14-2. However, the duration of immune response overtime after vaccination is inconclusive and may be associated with the risk of JE occurrence in adults. A cross-sectional study was conducted in 961 JE-vaccinated local residents aged 19-20 years in Beijing, China. 620 (65%) and 513 (53%) individuals were anti-JEV IgG antibody and neutralizing antibody (nAb) positive, respectively. The geometric mean titer (GMT) of nAb was 1:11, suggesting a seroprotection among the study population. As for IFN-γ production, peripheral blood mononuclear cell (PBMC) samples isolated from 60 subjects showed negative response following the stimulation with concentrated JEV particles. Overall, longer persistence of nAb response among vaccinees is observed than that of cellular immune response after 17-18 years of vaccination. Taken together, our results not only provide the data for evaluating herd immunity against JEV among vaccinated adults in Beijing but also offer useful information for JE prevention and control in endemic areas.

Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis.

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1-4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.

Cross neutralisation of Southeast Asian cobra and krait venoms by Indian polyvalent antivenoms.

Cross neutralisation of venoms by antivenom raised against closely-related species has been well documented. The spectrum of paraspecific protection of antivenom raised against Asiatic Naja and Bungarus (krait) venoms, however, has not been fully investigated. In this study, we examined the cross neutralisation of venoms from common Southeast Asian cobras and kraits by two widely used polyvalent antivenoms produced in India: Vins Polyvalent Antivenom (VPAV) and Bharat Polyvalent Antivenom (BPAV), using both in vitro and in vivo mouse protection assays. BPAV was only moderately effective against venoms of N. kaouthia (Thailand) and N. sumatrana, and either very weakly effective or totally ineffective against the other cobra and krait venoms. VPAV, on the other hand, neutralised effectively all the Southeast Asian Naja venoms tested, as well as N. naja, B. candidus and Ophiophagus hannah venoms, but the potency ranges from effective to weakly effective. In an in vivo rodent model, VPAV also neutralised the lethality of venoms from Asiatic Naja and B. candidus. In anesthetised rat studies, both antivenoms effectively protected against the N. kaouthia venom-induced cardio-respiratory depressant and neuromuscular blocking effects. Overall, our results suggest that VPAV could be used as alternative antivenom for the treatment of elapid envenomation in Southeast Asian regions including Malaysia, Thailand and certain regions of Indonesia.

Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine.

Live attenuated influenza vaccines (LAIVs) targeting seasonal influenza are produced in embryonated eggs and formulated as a trivalent preparation of three live attenuated vaccine strains, one A(H1N1) strain, one A(H3N2) strain and one type B strain. In this study, we describe an egg-based potency assay for estimating the 50% Egg infectious dose (EID50) of individual strains in the trivalent preparation by selective neutralisation of two strains and then estimating the infective titres of the non-neutralised strain. The test is highly specific, and no cross interference of heterologous antisera is observed in the estimation of individual titres. Individual strains with titres in the range of 6.5-7.0 log EID50 per 0.5 ml show intra-assay and inter-assay coefficients of variance ranging from 1.25% to 2.95%. This assay was developed to establish a simple, reliable and inexpensive egg-based assay for estimating the potency of individual strains in a trivalent preparation.

Hemagglutinin-pseudotyped green fluorescent protein-expressing influenza viruses for the detection of influenza virus neutralizing antibodies.

Influenza virus is a highly contagious virus that causes yearly epidemics and occasional pandemics of great consequence. Influenza virus neutralizing antibodies (NAbs) are promising prophylactic and therapeutic reagents. Detection of NAbs in serum samples is critical to evaluate the prevalence and spread of new virus strains. Here we describe the development of a simple, sensitive, specific, and safe screening assay for the rapid detection of NAbs against highly pathogenic influenza viruses under biosafety level 2 (BSL-2) conditions. This assay is based on the use of influenza viruses in which the hemagglutinin (HA) gene is replaced by a gene expressing green fluorescent protein (GFP). These GFP-expressing influenza viruses replicate to high titers in HA-expressing cell lines, but in non-HA-expressing cells, their replication is restricted to a single cycle.

Reproducibility of serologic assays for influenza virus A (H5N1).

Hemagglutination-inhibition (HI) and neutralization are used to evaluate vaccines against influenza virus A (H5N1); however, poor standardization leads to interlaboratory variation of results. A candidate antibody standard (07/150) was prepared from pooled plasma of persons given clade 1 A/Vietnam/1194/2004 vaccine. To test human and sheep antiserum, 15 laboratories used HI and neutralization and reassortant A/Vietnam/1194/2004, A/turkey/Turkey/1/2005 (clade 2.2), and A/Anhui/1/2005 (clade 2.3.4) viruses. Interlaboratory variation was observed for both assays, but when titers were expressed relative to 07/150, overall percentage geometric coefficient of variation for A/Vietnam/1194/2004 was reduced from 125% to 61% for HI and from 183% to 81% for neutralization. Lack of reduced variability to clade 2 antigens suggested the need for clade-specific standards. Sheep antiserum as a standard did not reliably reduce variability. The World Health Organization has established 07/150 as an international standard for antibody to clade 1 subtype H5 and has an assigned potency of 1,000 IU/ampoule.

Development of passive haemagglutination (PHA) and haemagglutination inhibition (HAI) technique for potency estimation of Cobra Antisnake Venom Serum (ASVS).

Antibodies against snake venom or antivenom potency are assayed quantitatively by in-vivo neutralization test in mice, which requires large number of laboratory animals. In potency assays of biological substances such as antivenoms, it is highly desirable to avoid suffering and death of animals by substituting in-vivo method with in-vitro methods, provided such methods measure life-saving capability with precision similar to that of in-vivo method. The in-vitro tests determine the neutralizing power of antivenom by permitting the evaluation of a particular biological activity of the venom and its neutralization after mixing the venom with the antivenom [Theakston RDG, Reid HA. Development of simple standard assay procedures for the characterization of snake venom. Bull WHO 1983;61:949-956; Gutierrez JM, Rojas G, Lomonte B, Gene JA, Chaves F, Alvarado J, et al. Standardizing of assays for testing the neutralizing ability of antivenoms. Toxicon 1990;28:1127-1129; Theakston R.D.G. Comments on letter of Gutierrez et al. on standardization of assays for testing the neutralizing ability of antivenoms. Toxicon 1990;28:1131-1132; Harvey AL, Barfaraz A, Thomson E, Faiz A, Preston S, Harris JB. Screening of snake venom for neurotoxic and myotoxic effects using simple in-vitro preparation from rodents and chicks. Toxicon 1994;32:257-265; World Health Organization Progress in characterization of venom and standardization of anti-venoms. Geneva: WHO offset publication; 1981. p. 58.]. Hence, the ideal requirements for an assay in detecting venom and venom antibody include high level of sensitivity, specificity (ability to differentiate between venom and venom antibody produced by closely related species of snakes), reproducibility and simplicity. A new in-vitro procedure for quantitative analysis of potency of ASVS by passive haemagglutination (PHA) and haemagglutination inhibition (HAI) has been explored. The methods described are simple, rapid, economical, reproducible and useful in replacing the more expensive in-vivo neutralization assays. Moreover, it also eliminates the use of laboratory animals.

The detection of anti-erythropoietin antibodies in human serum and plasma: part II. Validation of a semi-quantitative 3H-thymidine uptake assay for neutralizing antibodies.

Neutralizing antibodies to erythropoietin (EPO) can cause a loss of response to recombinant human EPO (rHuEPO) and lead to rare cases of sudden, unexplained, severe anemia in chronic renal failure patients treated with rHuEPO. An assay for neutralizing anti-EPO antibodies has been validated that is based on the inhibition of proliferation of human UT-7/EPO cells, an immortalized cell line, by neutralizing antibodies in serum test samples using 3H-thymidine as a marker for proliferation. The dependence of the human cell line on EPO for growth and proliferation in a concentration-dependent manner enabled the validation of a rHuEPO standard curve for cell proliferation that can be used to determine the presence of neutralizing anti-EPO antibodies in serum samples. Proliferation of the cells increases with increasing concentrations of EPO, forming an S-shaped standard curve, which is fit with a 4-parameter logistic model, between 2.5 and 50 mU/mL rHuEPO, with a percent coefficient of variation (% CV) from 8.7% to 22.1% and a % accuracy of 103.5% to 109.5%. Anti-EPO antibodies and serum with anti-EPO antibodies neutralize UT-7/EPO proliferation by 10 mU/mL rHuEPO in a concentration- or dilution-dependent manner with < or = 25% CV. Percent neutralization is calculated by determining the amount of EPO recovered from the original 10 mU/mL added using the formula [((10-concentration recovered)/10)x100%]. Stem cell factor (SCF) stimulated cell proliferation, but not as effectively as rHuEPO. Antibodies to SCF were not able to inhibit the proliferative response induced by EPO and vice versa, confirming the specificity of the assay for antibodies to EPO. High EPO levels can impact both the radioimmunoprecipitation and neutralization assays to produce a false negative result. However, the impact can be mitigated by the large dilutions used in the neutralization assay.

Yellow fever 17D vaccine safety and immunogenicity in the elderly.

The incidence of serious and severe multisystem adverse events (AEs) following yellow fever (YF) 17D vaccine is higher in persons of advanced age. One hypothesis for the occurrence of these AEs in the elderly is immunological senescence and a reduced ability to clear the vaccine virus infection. We determined age-specific rates of serious and nonserious AEs in two large clinical trials of two YF 17D vaccines from different manufacturers. In addition, we analyzed AEs reported in a large general practice data base in the United Kingdom. Neutralizing antibody responses were compared in young and elderly subjects. In the clinical trials, involving a total of 4,532 subjects, there were no neurological and viscerotropic AEs; interestingly, the incidence of common injection site and systemic AEs was significantly lower in elderly than in younger subjects. The neutralizing antibody categorical and quantitative responses were equivalent across younger and elderly subjects. In contrast, the larger retrospective analysis of 43,555 persons receiving YF 17D in the UK general practice database revealed a higher incidence of significant neurologic and multisystem AEs with advancing age. The age-specific reporting rate ratio (RRR) was approximately twice that in the 25-44 year-old reference group for subjects in the 45-64 year age group (RRR 1.82; 95% CI 0.88,3.77) and 3-fold higher for the 65-74 year-old age group (RRR 2.82; 95% CI 0.81, 9.81). These results are consistent with previous reports on YF vaccine safety in the US (Martin M, et al. Emerg Infect Dis 2001;6:945-51; Khromova et al., Vaccine 2005;23:3256-63). In elderly persons, YF 17D vaccine is associated with a higher frequency of significant AEs in the elderly but a lower incidence of common nonserious side-effects. The neutralizing antibody response, which is the mediator of protective immunity to YF, is not diminished in healthy, elderly persons.