Vaccine 27 (2009) 3501–3504
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Letter to the Editor
Prevalence of serum neutralizing antibodies against chimpanzee adenovirus 63 and human adenovirus 5 in Kenyan Children, in thecontext of vaccine vector efficacy
a r t i c l e i n f o
Keywords:Simian adenoviral vectorMalaria vaccineVector neutralizing antibodies
a b s t r a c t
Vaccination against Plasmodium falciparum malaria could reduce the worldwide burden of this disease,and decrease its high mortality in children. Replication-defective recombinant adenovirus vectors car-rying P. falciparum epitopes may be useful as part of a vaccine that raises cellular immunity to thepre-erythrocytic stage of malaria infection. However, existing immunity to the adenovirus vector resultsin antibody-mediated neutralization of the vaccine vector, and reduced vaccine immunogenicity. Our aimwas to examine a population of children who are at risk from P. falciparum malaria for neutralizing immu-nity to replication-deficient recombinant chimpanzee adenovirus 63 vector (AdC63), compared to humanadenovirus 5 vector (AdHu5). We measured 50% and 90% vector neutralization titers in 200 individual
sera, taken from a cohort of children from Kenya, using a secreted alkaline phosphatase neutralizationassay. We found that 23% of the children (aged 1–6 years) had high-titer neutralizing antibodies to AdHu5,and 4% had high-titer neutralizing antibodies to AdC63. Immunity to both vectors was age-dependent.Low-level neutralization of AdC63 was significantly less frequent than AdHu5 neutralization at the 90%neutralization level. We conclude that AdC63 may be a useful vector as part of a prime-boost malaria1
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. Introduction
Plasmodium falciparum causes clinical malaria in up to 650 mil-ion people annually [1], one million children die from P. falciparum
alaria every year [2]. A vaccine against malaria could reduce theurden of disease, alongside other strategies.
Cellular immunity induced by a vaccine could protect againsthe pre-erythrocytic stages of Plasmodium infection. Irradiated P.alciparum sporozoites administered to human volunteers can pro-ect against malaria following experimental challenge for 6 months3]. In mice, the immunity generated by irradiated sporozoitess dependent on CD8 + T cell activity: non-immunized mice cane completely protected from infection with P. yoelii sporozoitesy adoptive transfer of thrombospondin related adhesion proteinTRAP)-specific CD8 + T lymphocytes [4]. In humans poxvirus vac-ines encoding ME-TRAP have reduced parasite burdens in the liversf sporozoite challenged vaccinees by around 90% [5].
Adenovirus recombinant vectors could be safe and immuno-enic delivery vehicles for a malaria vaccine. Adenovirus-basedaccines have been shown to induce a strong cytolytic T lympho-yte response to the transgenic antigen, and can protect completelygainst infection in a mouse model [6].
The efficacy of adenovirus-based vaccines at generating CD8 + Tell immunity to a transgene [7–11], and protecting from infection12,13], is lower in subjects with existing humoral immunity to theector serotype. Neutralizing antibodies decrease the expression of
he transgene carried by the vector [7], and may be responsible for,s well as predictive of, decreased immunogenicity. Neutralizingntibodies to Human Adenovirus 5 (AdHu5), a common vaccineector candidate, have an adult prevalence between 30% and 50%264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.03.080
© 2009 Elsevier Ltd. All rights reserved.
in the USA and Western Europe, and up to 98% in surveyed Africancountries [14–17]. Normal exposure to human adenoviruses leadsto a rapid increase in neutralizing antibody prevalence duringchildhood [18]. In the recent STEP Study CD8 + T lymphocyteresponses to an AdHu5 vector-based HIV-1 vaccine were sig-nificantly lower in human volunteers showing serum antibodyimmunity to AdHu5 [25].
Previous studies suggest that the prevalence of neutralizing anti-bodies to chimpanzee adenoviruses may be very low in targetpopulations [17–21]. Chimpanzee adenovirus vectors, like humanadenovirus vectors, can generate CD8 + T cell immunity to trans-genic peptides [19]. Chimpanzee adenovirus 63 (AdC63) has beenused to create a replication-deficient, ME-TRAP transgenic P. falci-parum malaria vaccine, currently in Phase I clinical testing as thefirst simian virus to be assessed as a vaccine vector in humans.
The object of this study was to quantify the prevalence ofneutralizing antibodies to AdHu5 and AdC63 in a population ofchildren likely to benefit from a malaria vaccine. Children are at thegreatest risk from P. falciparum malaria; the sample was of childrenaged from 1 to 6 years from Kilifi, Kenya. The virus neutralizationtiters in this group would be informative as to the clinical scopeof a vaccine based on the AdC63 vector. Here we report levels ofneutralizing antibodies against AdC63 in a target population formalaria vaccination.
2. Materials and methods
2.1. Viruses
Replication-deficient adenovirus transgenic E1-deficient vec-tors expressing secreted alkaline phosphatase (SEAP), AdHu5–SEAP
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502 Letter to the Editor / Va
nd AdC63–SEAP, were obtained from Okairòs, Italy. The transgeniciruses share penton, hexon and fibre coding regions with vaccineectors and wild-type viruses of the same serotype.
The ratio of virus particles per cell used in the neutralizationssay was calibrated to fall within the linear range of luminescencentensity when the assay was performed [20].
.2. Human serum samples
Two hundred serum samples were used from a cohort collectedor pre-vaccination screening. These samples were obtained fromealthy children aged from 1 to 6 years, living in villages aroundilifi, Eastern Kenya. Data relating to age, sex and residence of thearticipants was available for 199 of these 200 samples.
Ethical approval was obtained from the Kenyan Medicalesearch Council (KEMRI) National Ethics Committee and Oxford-hire Research Ethics Committee.
Sera were stored at −80 ◦C and inactivated at 56 ◦C for 90 minrior to use.
.3. Virus neutralization assays
Adenovirus-specific neutralizing antibody (NAb) titers weressessed using a SEAP quantitation assay as described previously21].
HEK293A cells were infected with AdHu5–SEAP or AdC63–SEAP.ecombinant adenoviruses were incubated with five serial dilu-ions of serum in FBS–DMEM before infection. The final serumilutions were 1:18, 1:72, 1:288, 1:1152, 1:4608; each serum sam-le was tested in duplicate. The antibody-containing fraction (FBSnd human serum) was 11%.
Supernatants were collected and assayed for SEAP concentrationsing CSPD (Tropix) according to the manufacturer’s instructions.uminescence intensity was measured using a MicroBeta Trilux450 luminometer (PerkinElmer); Luminescence intensity (RLU)as standardized against that of uninfected wells. SEAP concen-
ration was calculated from the standard relation between SEAPoncentration and RLU, measured with each set of assays.
Neutralization titers were defined as the serum dilution at whichEAP concentration was 50% that in wells infected with virus alone.0% neutralization titers were also calculated. Neutralization titeras calculated by linear interpolation of adjacent values.
.4. Statistical analysis
To evaluate the difference in neutralization titer prevalence inroups of children, a Wilcoxon test was used. Prevalence of specificiters was assumed to be binomially distributed in the population;5% confidence intervals were calculated using the Exact methodor binomial distributions. The cohort was stratified into two ageroups, allowing comparison between younger and older children.
. Results
We first evaluated the prevalence of neutralizing antibodiesgainst AdHu5–SEAP and AdC63–SEAP in sera taken from 200hildren aged from 1 to 6 years. These children were resident inunju sub-location, in Kilifi district on the Kenyan coast—and sub-equently participated in a trial of a poxvirus prime-boost malariaaccine [22].
Of the 200 children, 23% (95% confidence interval 17.4–29.5%)
ere found to have high titer AdHu5 vector-specific neutralizingntibody (i.e. titers in excess of 200), compared with 4% (1.7–7.7%)gainst the AdC63 vector (p < 0.0001). Levels of very high-titer neu-ralizing antibodies (titers in excess of 1000) were also greater fordHu5 vector (9%; 5.4–13.9%) than for AdC63 vector (0.5%; 0–2.8%).
Fig. 1. Neutralization titer in children aged 1–2 years. Neutralization titer in childrenaged 3–6 years.
Serum NAb levels are shown for two age groups in Fig. 1. Onaverage, titers against AdC63 were lower than against AdHu5 inthe cohort studies. The geometric mean titer against AdC63 (15.9;13.8–18.0) was found to be significantly different from AdHu5 (35.3;33.1–37.6). In contrast to the findings for high titer antibodies, lowtiter antibodies at the 50% neutralization level were not found tobe more common against AdHu5 than for AdC63 (14% vs 17.5%;p = 0.41).
90% neutralization titres were also tested. There was a sig-nificant difference between the incidence of high neutralizationtitres against AdHu5 (12.5%; 7.3–19.5%) and AdC63 (one child; 0.8%;0–4.0%) in the 3–6 years age group. No children had any detectableantibody neutralization at this level against AdC63 in the 1–2 yearsage group (confidence interval 0–4.0%); seven (9.7%; 4.0–19.0%)children in the same age group had detectable antibodies againstAdHu5. The difference in high neutralization titer incidence was notsignificant in this age group. Low antibody titers at the 90% neutral-ization level were found to be significantly higher against AdHu5than against AdC63 (15% vs 2%; p < 0.001).
4. Discussion
Chimpanzee adenovirus 63 could serve as a vector for childhoodimmunization against malaria, and overcome the problems associ-ated with existing immunity to human adenoviruses [13]. There areno previous reports regarding exposure to AdC63. Our report is con-cerned with antibody neutralization in a small cohort of childrenfrom one malaria-endemic area in Africa; however, our findings
for AdHu5 neutralization are consistent with reports from otherregions. The prevalence of high-titer serum neutralization of AdHu5in the two age groups considered in our cohort was found to be sim-ilar to those in a cohort of children from South Africa investigatedby Thorner et al. [18]. In this sample from Kilifi, Kenya, 8.3% of the
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Letter to the Editor / Va
hildren aged 1–2 years had high neutralization titers to AdHu5i.e. >200), compared to 12.8% in the South African cohort of theame age. The subsequent age groups (3–6 years in this Kenyanample and 2–7 years in the South African cohort) had 31.3% preva-ence and 38.5% prevalence of high AdHu5 neutralization titers,espectively.
We observed a very low prevalence of high-titer serum neutral-zation against AdC63 when compared to AdHu5 in all 200 childrenested, and this difference was particularly marked among childrenged 3–6 years. This probably reflects the acquisition of immunityo human adenoviruses with age, as they circulate in the popu-ation. The low prevalence of AdC63 neutralization in both ageroups, if representative of other populations in malaria endemicreas, could indicate greater flexibility in the use of an AdC63-ased vaccine in children of different ages, so prime-boost regimensith peak immunogenicity and antimalarial efficacy could be cho-
en. High-titer serum neutralization is clinically significant, as thisan reduce the effective number of vaccine carriers by the orderf 100 times [7]. CD8 + T cell vaccine responses are decreased inhe presence of vector-reactive CD8 + T cells, which correlate witheutralizing antibodies in humans [24–26]. It is unclear whetherhere is widespread CD8 + T cell interference between heterolo-ous adenovirus vectors; studies of CD8 + T cell adenovirus vaccinemmunogenicity have shown marginally decreased responses fol-owing heterologous vector immunization [7,9] though othersave not observed this effect [14,15]. Heterologous CD8 + T cell
nterference appears to have little impact on adenovirus vectormmunogenicity.
Low neutralization titers observed depended on the neutraliza-ion level tested; In this study, we considered 50% neutralizationiters, which are more sensitive to low-level neutralization, and0% neutralization titers, which are more specific. Low AdHu5eutralization titres at the 90% level (15.0%; 10.4–20.7%) are sim-
lar to those observed by Thorner et al. at the 90% neutralizationevel, in similar age groups (15.4%) [19]. AdC63 low-titer neu-ralization was significantly lower across the two age groups2.0%; 0.55–5.0%).
Neutralization of AdC63 could result from cross-reactivity ofther serum antibodies to this vector. Circulation of AdC63 inumans has not been documented, but cannot be ruled out. Stud-
es of other chimpanzee adenoviruses yielded very low levels oferum neutralization in other populations tested [17–21]. In theSA, neutralizing antibody titers against Chimpanzee Adenovirus8 in healthy volunteers were measured at 2% [17]; in Thailand,igh neutralization titers against chimpanzee adenovirus serotypesand 1 were 1% and 2%, respectively [20]. In Cameroon, Nigeria andote d’Ivoire, all countries with a holoendemic malaria incidence1], the adult prevalence of antibodies against two chimpanzeedenoviruses did not exceed 10% [23]. It is possible that AdC63 isartially neutralized by antibodies against other adenoviruses, orgainst another pathogen.
The results of this study in a small cohort of children from aalaria-endemic area confirm the findings of previous studies on
he exposure and serum neutralization of AdHu5 in children, androvide new findings about chimpanzee adenovirus immunity inumans. The low prevalence of high titer neutralizing antibodies todC63 in both target age groups in our cohort is very encouraging
or its potential use as a malaria vaccine vector.
cknowledgements
This work was supported by the Wellcome Trust. AVSH is aellcome Principal Research Fellow. This paper is published with
ermission of the director of the Kenyan Medical Research InstituteKEMRI).
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E-mail address: [email protected](M. Dudareva)
5 October 2008Available online 16 April 2009
504 Letter to the Editor / Va
M. Dudareva a,∗,L. Andrews a,S.C. Gilbert a,
P. Bejon b,K. Marsh b,
J. Mwacharo b,O. Kai b,
A. Nicosia c,A.V.S. Hill a
a Jenner Institute Laboratories, Old Road Campus,
University of Oxford, Oxford OX3 7LF, United Kingdomb Kenya Medical Research Institute, Centre forGeographical Medicine Research (Coast), Kenya
c Okairòs S.r.l., 22 Via Castelli Romani,Pomezia, 00040 Rome, Italy
27 (2009) 3501–3504
∗ Corresponding author:Tel.: +44 1865741976.
