Persistence of Antibody After a Vi-Tetanus Toxoid Conjugate Vaccine and Effect of Boosting With a Plain Polysaccharide Vaccine on Vi Antibody and Antigen-Specific B Cells

Abstract

Background
Salmonella enterica serovar Typhi is estimated to cause 9 to 13 million cases of typhoid fever annually. Typhoid conjugate vaccines represent a promising prophylactic measure to prevent disease, but there are few data assessing persistence of immunity. The effect of a Vi polysaccharide booster vaccine in individuals previously vaccinated with the Vi-tetanus toxoid typhoid conjugate vaccine has not been assessed previously.

Methods
Thirty five healthy adult volunteers received a single dose of the Vi conjugate vaccine (Vi-TT) and 37 received a single dose of Vi polysaccharide vaccine (Vi-PS) prior to oral challenge with live S . Typhi bacteria as part of a randomised controlled, phase 2b study. In addition to data previously published showing persistence of Vi IgG and IgA antibodies for 7 months after Vi vaccination, titres were measured at intervals until 13 months post-vaccination. Ten participants who received Vi-TT (both challenged and unchallenged) were re-vaccinated with Vi-PS at an interval of 19-23 months post-prime. Anti-Vi IgG and IgA titres, and Vi-specific antibody secreting cells and memory B cells were measured at seven days and one month post-boost.

Findings
Vi IgG and IgA antibody titres remained significantly elevated above baseline levels 13 months after priming with Vi-TT, with a 4-fold rise retained in 90% and 88% of recipients (Vi IgG and IgA, respectively). Anti-Vi IgG and IgA antibody titres were found to persist at higher levels in participants who received a single dose of Vi-TT than in those who received Vi-PS. No significant boost in Vi-antibody titre was observed in response to oral challenge with S. Typhi bacteria, one month after vaccination. Following a Vi-PS booster vaccination in those previously vaccinated with Vi-TT, anti-Vi IgG and IgA titres were significantly elevated, with similar titres observed at one month post-boost compared with one month after primary vaccination. The frequency of Vi-specific IgA antibody secreting cells increased significantly 7 days post-boost compared with pre-boost. No memory B cell response was observed following Vi-PS booster vaccination.

Interpretation
Strong persistence of anti-Vi IgG and IgA following Vi-TT vaccination suggests that the conjugate vaccine may offer durable protection, supporting its use in endemic settings.

Full text

Persistence of Antibody After a
Vi-Tetanus Toxoid Conjugate
Vaccine and Effect of Boosting
With a Plain Polysaccharide
Vaccine on Vi Antibody and
Antigen-Specific B Cells
Thomas Bentley
1,2†
, Elizabeth Jones
1,2
*
†
, Celina Jin
1,2
, Maria Moore
1,2
,
Jonathon Gardner
1,2
, Jennifer Hill
1,2‡
and Andrew J. Pollard
1,2‡
1
Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, United Kingdom,
2
The National Institute for
Health Research Oxford, Biomedical Research Centre, Oxford, United Kingdom
Background: Salmonella enterica serovar Typhi is estimated to cause 9 to 13 million cases
of typhoid fever annually. Typhoid conjugate vaccines represent a promising prophylactic
measure to prevent disease, but there are few data assessing persistence of immunity. The
effect of a Vi polysaccharide booster vaccine in individuals previously vaccinated with the Vi-
tetanus toxoid typhoid conjugate vaccine has not been assessed previously.
Methods: Thirty five healthy adult volunteers received a single dose of the Vi conjugate
vaccine (Vi-TT) and 37 received a single dose of Vi polysaccharide vaccine (Vi-PS) prior to
oral challenge with live S. Typhi bacteria as part of a randomised controlled, phase 2b
study. In addition to data previously published showing persistence of Vi IgG and IgA
antibodies for 7 months after Vi vaccination, titres were measured at intervals until 13
months post-vaccination. Ten participants who received Vi-TT (both challenged and
unchallenged) were re-vaccinated with Vi-PS at an interval of 19-23 months post-prime.
Anti-Vi IgG and IgA titres, and Vi-specific antibody secreting cells and memory B cells
were measured at seven days and one month post-boost.
Findings: Vi IgG and IgA antibody titres remained significantly elevated above baseline
levels 13 months after priming with Vi-TT, with a 4-fold rise retained in 90% and 88% of
recipients (Vi IgG and IgA, respectively). Anti-Vi IgG and IgA antibody titres were found to
persist at higher levels in participants who received a single dose of Vi-TT than in those
who received Vi-PS. No significant boost in Vi-antibody titre was observed in response to
oral challenge with S. Typhi bacteria, one month after vaccination. Following a Vi-PS
booster vaccination in those previously vaccinated with Vi-TT, anti-Vi IgG and IgA titres
were significantly elevated, with similar titres observed at one month post-boost
compared with one month after primary vaccination. The frequency of Vi-specific IgA
Frontiers in Tropical Diseases | www.frontiersin.org September 2021 | Volume 2 | Article 7097451
Edited by:
Jean-Louis Excler,
International Vaccine Institute,
South Korea
Reviewed by:
Yun-Chi Chen,
Morgan State University, United States
Tejram Sahu,
Johns Hopkins University,
United States
*Correspondence:
Elizabeth Jones
elizabeth.jones@paediatrics.ox.ac.uk
†
These authors have contributed
equally to this work
‡
These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Vaccines for Tropical Diseases,
a section of the journal
Frontiers in Tropical Diseases
Received: 14 May 2021
Accepted: 30 August 2021
Published: 20 September 2021
Citation:
Bentley T, Jones E, Jin C, Moore M,
Gardner J, Hill J and Pollard AJ (2021)
Persistence of Antibody After a Vi-
Tetanus Toxoid Conjugate Vaccine
and Effect of Boosting With a Plain
Polysaccharide Vaccine on Vi Antibody
and Antigen-Specific B Cells.
Front. Trop. Dis. 2:709745.
doi: 10.3389/fitd.2021.709745
ORIGINAL RESEARCH
published: 20 September 2021
doi: 10.3389/fitd.2021.709745

antibody secreting cells increased significantly 7 days post-boost compared with pre-
boost. No memory B cell response was observed following Vi-PS booster vaccination.
Interpretation: Strong persistence of anti-Vi IgG and IgA following Vi-TT vaccination
suggests that the conjugate vaccine may offer durable protection, supporting its use in
endemic settings.
Keywords: typhoid fever, enteric fever, Salmonella typhi (S. typhi), vaccination, humoral immunity, Vi vaccine
INTRODUCTION
Despite increasing access to improved water sources globally,
typhoid fever continues to be prevalent in low and lower middle
income countries (LMIC), with an estimated 9 to 13 million cases
and 145,000 to 161,000 deaths annually (1). India and South-East
Asia suffer the highest infection rates, and there are frequent
epidemics in sub-Saharan Africa, with severity of illness and
mortality highest in early childhood (2–4).
Efforts to control and prevent typhoid fever have generally
included a combination of public health measures, health
education and vaccination programmes. Although antibiotics
reduce the fatality rate of patients with an acute infection to
around 1%, they have little impact on disease incidence, and
improper use of antibiotics can drive the emergence of antibiotic
resistant strains (5,6). Vaccination offers a prophylactic option that
may be quicker and more cost effective to implement in the short
term than infrastructure improvements. Vaccination against S.
Typhi can provide the population with herd immunity, helping to
protect those who are vulnerable to disease or cannot safely receive
vaccines such as immunocompromised individuals (7,8).
Furthermore, evidence from human typhoid challenge studies
suggests that an effective vaccination schedule may reduce onward
transmission of S. Typhi by reducing bacterial shedding in stool (9).
Polysaccharide vaccines elicit an antibody response independently
of T cells. In the absence of memory B cell formation further doses of
vaccine are recommended every 2-3 years (10,11). Furthermore,
polysaccharide vaccines are non-immunogenic in infants; as the
ability to respond to polysaccharide antigens develops alongside the
maturation of the splenic marginal zone at around 18 to 24 months
of age.
Conjugate vaccines utilise a protein carrier to enhance the
response to polysaccharide antigen by inducing T cell help and
germinal centre formation which results in memory B cell
production. Conjugate vaccines have been demonstrated to be
efficacious in preventing numerous infections caused by
encapsulated pathogens including Haemophilus influenzae type
b, pneumococcal infection and meningococcal disease (12–14).
Immunogenicity has been demonstrated in infants for whom
vaccines were previously unavailable, and higher antibody
responses have been elicited in older recipients (15–18). A
novel typhoid tetanus toxoid vaccine (Vi-TT) was shown to be
efficacious in adults in an established controlled human infection
model (CHIM), as well as demonstrating a protective efficacy of
81.6% after one year in children aged 9 months to 16 years in
Nepal, in a phase three randomised controlled trial (19,20).
There is mounting evidence that typhoid conjugate vaccines
(TCV) are safe, efficacious and highly immunogenic Several
TCVs are now prequalified by the WHO and are being rolled
out in in endemic countries, however data on persistence of
antibodies and duration of protection is unknown (21,22).
The Vaccines Against Salmonella Typhi (VAST) trial was
initiated in 2015 to evaluate the protective efficacies of a typhoid
conjugate vaccine (Vi-TT) and the licensed capsular
polysaccharide vaccine (Vi-PS) using a CHIM. The 2017
publication of this study reported a protective efficacy for Vi-
TT of 55%. Of note, higher Vi IgG titres were elicited in the Vi-
TT group at one month after vaccination when compared with
Vi-PS vaccinees (19). An extension to the main study, in which
participants primed with Vi-TT were later boosted with Vi-PS,
was added as part of a project developed to produce high affinity
Vi-specific monoclonal antibodies for use as research tools. Here
we describe the persistence of Vi antibodies after a single dose of
Vi-TT or Vi-PS vaccine, and assess the effect of polysaccharide
booster vaccination on antibody titre, antibody secreting cell
(ASC) response and memory B cell production in participants
primed with Vi conjugate vaccine.
METHODS
Study Design and Participants
Samples were obtained from volunteers participating in an
observer and participant-masked, randomised, controlled,
phase 2b clinical trial, evaluating protection from typhoid
infection following Vi-TT vaccination using a S. Typhi CHIM
(group A) (19). Volunteers in group A were invited to participate
in a booster study, along with volunteers from a separate cohort
randomised to receive Vi-PS or Vi-TT without subsequent oral
challenge as part of a study to develop a Vi IgG serum standard
(group B) (23). Five participants were recruited from both group
A and group B to form the booster study participants (group C).
Details of these groups are presented in Table 1 and Figure 1.
Group C participants received a Vi-PS booster vaccination
(Typhim Vi™, SanofiPasteur).
Quantification of Vi Antibodies
Serum Vi-specific IgG quantification was performed using a
commercial enzyme-linked immunosorbent assay (ELISA) kit
(VaccZyme™Human Anti Salmonella Typhi Vi IgG Enzyme
Immunoassay Kit, Binding Site, Birmingham UK), as per the
manufacturer’s instructions. Values below the lower limit of
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assay sensitivity (7.4 EU/ml) were assigned a value of 3.7 EU/ml.
For the quantification of serum Vi IgA antibodies the kit was
modified to include a sheep anti-human IgA secondary antibody
(Binding Site, Birmingham UK) prepared 1:12,000 in 1 x
phosphate buffered saline, 10% foetal bovine serum. Values
below the lower limit of assay sensitivity (3.125 EU/ml) were
assigned a value of 1.56 EU/ml.
Antibodies were quantified in serum samples collected 13
months post primary vaccination for group A only [data for
group A PV, 1, 2, 4 and 7 months are published previously (24)],
and for all participants recruited into group C prior to boost
vaccination (PV Booster), seven days post-boost and 1 month
post-boost.
Detection of Plasmablasts
Ex vivo enzyme linked immunosorbent spot (ELISpot) assays
were used to detect Vi-specific antibody secreting cells (ASC) for
participants in group C prior to booster vaccination (PV
Booster), and at 7 and 28 days post-boost. Peripheral Blood
Mononuclear Cells (PBMCs) were isolated from fresh whole
blood within 6 hours of collection, 2.5 x 10
5
cells/well were
seeded on to ELISpot plates (MultiScreen-HA Filter Plates,
Millipore, MAHAS510) pre-coated with Vi antigen (12/244,
NIBSC) at 10µg/mL. Cells were incubated overnight (16-24
hours) at 37°C and 5% CO
2
before development with alkaline
phosphatase conjugated secondary antibodies (goat anti-human
IgG, and IgM or IgA, Calbiochem) and a substrate development
kit (Bio-rad laboratories ltd). Wells pre-coated with pan goat
anti-human immunoglobulin, or PBS were used as positive and
negative controls, respectively. The results are expressed as ASCs
per million PBMCs, the range of the assay was 3 –141 Vi specific
ASCs per million PBMCs.
Detection of Memory B Cells
ELISpot was used to detect Vi specific memory B cells from
cultured stimulated PBMCs for participants in group C at pre
vaccination, and at 7 and 28 days post vaccination. Briefly, frozen
PBMCs were thawed and stimulated for 5 days with an antigen
cocktail containing Staphylococcus aureus Cowans Strain –
Pansorbin cells (1:5000, VWR International Ltd), CpG-ODN
2006 (1.7 µg/mL, Source BioScience UK), and Pokeweed
Mitogen (83.33ng/mL, Sigma). After 5 days the cells were
harvested, washed and counted. The ELISpot assay was then
performed as described above. Some wells were also pre-coated
with pan goat anti-human immunoglobulin or PBS as positive
and negative controls. All samples included in the analysis had
positive IgG producing plasmablasts after memory cells culture
(10/10).
Statistical Analysis
The data were analysed using GraphPad Prism 8.0.0. Mann-
Whitney U tests were used to compare time points between
vaccine arms, with a one-way ANOVA Kruskal-Wallis test for
multiple comparisons. A Wilcoxon test was used to compare
time points within vaccine arms, with a Friedman test adjusting
for multiple comparisons. Comparisons between vaccines arms
were treated as unpaired, whilst longitudinal comparisons were
treated as paired. A p-value of <0.05 was considered to be
statistically significant.
Role of the Funding Source
The funders of the study had no role in study design, data
collection, data analysis, data interpretation, or writing of
the report.
RESULTS
Persistence of Vi IgG and IgA in Response
to a Single Vi-TT or Vi-PS Vaccination
Vi IgG and IgA titres remained significantly elevated above pre-
vaccination levels in both group A vaccine arms 13 months after
prime,andinthegroupAVi-TTrecipients19–23 months after
prime (median 20 months, n=5) (Vi-PS not measured) (Figure 2). A
significant reduction in anti-Vi IgA titre was observed in both vaccine
arms, and for anti-Vi IgG in Vi-TT vaccine recipients, between one
month post-prime and 13 months post-prime (Figure 2).
TABLE 1 | Participant groups in the Vaccines Against Salmonella Typhi trial from which samples were taken for analysis in this study.
Group Composition Vaccine administered Post-vaccine procedures
A 112 participants with no
previous typhoid vaccination
or residency of >6 months in
a typhoid endemic area
Randomised 1:1:1 to
receive a single dose of
either:
•Vi tetanus toxoid
conjugate (Vi-TT),
•Vi polysaccharide (Vi-
PS), or
•MenACWY control
vaccine
After 28 days participants were orally challenged with 1-5 x 10
4
CFU S. Typhi and monitored for
2 weeks for development of typhoid infection (persistent fever ≥38°C for 12 or more hours) or S.
Typhi bacteraemia. Antibiotics were commenced at the time of typhoid fever diagnosis or day 14
post-challenge in those who were not diagnosed with typhoid fever.
B 30 participants Received either Vi-TT
(n=15) or Vi-PS (n=15)
Immunogenicity analysis, and serum donation for production of the first international Vi IgG
serum standard (16/138, NIBSC, Potters Bar)
C 10 participants primed with
Vi-TT (comprised of 5
individuals from group A and
5 from group B)
Vi-PS vaccination at 19
to 23 months after
administration of Vi-TT
(median interval 20
months)
Immunogenicity analysis only
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90% (26/29) of participants vaccinated with Vi-TT and 85% (23/27)
of participants vaccinated with Vi-PS retained an IgG titre at least
four times that of the PV titre 13 months after vaccination. 88% (23/
26) of participants vaccinated with Vi-TT and 77% (20/26) of
participants vaccinated with Vi-PS retained an IgA titre at least
four times that of the PV titre 13 months after vaccination. In all five
Vi-TT recipients in group A subsequently recruited into group C,
anti-Vi IgG and IgA titres measured at 20 months post-prime
demonstrated antibody titres four-fold greater than baseline.
Participants from group B were not included in the antibody
persistence analysis as some volunteers in this group had
previously received typhoid vaccines outside of the trial setting.
FIGURE 2 | Vi IgG and Vi IgA antibody produced in response to Vi-TT vaccination is significantly elevated over baseline levels beyond 1 year. Vi antibody titres were
determined in naïve participants (group A) by ELISA at 13 months post-vaccination (n = 72), and at time points between 19 and 23 months post-vaccination in group
C participants (n=5) (shown plotted at the median of 20 months to aid visualisation). To provide context for these data, Vi antibody titres measured prior to
vaccination (PV) and at 1, 2, 4, and 7 months post-vaccination are also plotted [published previously (24)]. Comparisons between vaccine arms Vi-PS (grey) and Vi-
TT (black), and between time points were performed. Adjusted p-values are indicated. *p ≤0.05; **p ≤0.01; ***p ≤0.001; ****p ≤0.0001. Vertical lines demarcate
time points.
FIGURE 1 | Trial profile. Vi-TT, Vi-tetanus toxoid conjugate vaccine; Vi-PS, Vi-polysaccharide vaccine; V2, study visit 4 - 6 weeks post-vaccination.
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Vi IgG and IgA Responses to Vi-PS
Booster Vaccination
Ten participants primed with Vi-TT were boosted with Vi-PS.
Demographic information for participants who received a
booster vaccination is presented in Supplementary Table 1.
Five were from the group A challenge study (three diagnosed
with typhoid within 14 days of receiving challenge, and two non-
diagnosed), and five from the group B non-challenged cohort. In
recipients of a Vi-TT primary vaccine, booster vaccination with
Vi-PS induced a significant increase in both anti-Vi IgG and IgA
antibody titre at one month (Figure 3).
Anti-Vi IgG titres were comparable at one month post Vi-TT
prime and one month post Vi-PS boost (Figure 4). Anti-Vi IgG
titres prior to boost were significantly higher than pre-prime
titres, as such the Vi-PS booster vaccination generated a smaller
fold rise above pre-boost titres after one month as compared with
primary vaccine response. Correlation of pre-boost anti-Vi IgG
and fold change in anti-Vi IgG (pre-boost to day 28 post-boost)
demonstrated a strong negative relationship (Spearman’s
correlation coefficient = -0.90, p = 0.0008) (Figure 5). A
negative relationship between titre prior to boost and the fold
change in titre generated by boost was also observed for anti-Vi
IgA (Spearman’s correlation coefficient = -0.61, p = 0.064).
Effect of S. Typhi Challenge on Anti-Vi
IgG Titre
To assess the effect of exposure to S. Typhi on anti-Vi IgG titres
in participants immunised with Vi-TT, the decay in antibody at
one and 20 months post primary Vi-TT vaccination was
compared between individuals who underwent S. Typhi
challenge (n=5), and those who were not exposed (n=5). A
Mann-Whitney test comparing fold change between these two
groups showed no significant difference (p=0.42), suggesting that
a single oral exposure to S. Typhi has no effect on the persistence
of serum anti-Vi IgG.
Vi IgG, IgA, and IgM Peripheral ASC
Responses to a Vi-PS Booster Vaccination
Vi-PS booster vaccination induced a significant increase in the
frequency of Vi IgA ASCs between pre-boost (median = 3.0
ASCs/10
6
PBMCs) and 7 days post-boost (median = 7.7 ASCs/
10
6
PBMCs), while no significant change was observed for ASCs
producing anti-Vi IgG or IgM (Figure 6). In contrast, the initial
Vi-TT vaccination induced significant increases in both Vi
specific IgG and IgM ASCs (25). No data were collected on the
anti-Vi IgA ASC response to primary conjugate vaccination.
FIGURE 3 | A booster Vi-PS vaccination in participants primed with Vi-TT induced a significant increase in Vi IgG and Vi IgA antibody titre by D28. Vi antibody titres
were determined by ELISA prior to Vi-PS vaccination (PV) in individuals primed with Vi-TT, and at 7 and 28-days post-boost, and comparison of titres between time
points was performed (n= 10). Adjusted p-values are indicated. **p ≤0.01; ***p ≤0.001.
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Vi IgG and IgA Circulating Memory B Cell
Response to a Vi-PS Booster Vaccination
No significant change in the frequencies of Vi-specific IgG or IgA
memory B cells were observed seven and 28 days after Vi-PS
booster vaccination (Figure 7).
DISCUSSION
In this study we describe the persistence of the Vi antibody
response to plain Vi polysaccharide and Vi tetanus toxoid
conjugate vaccines, showing a similar kinetic profile with a
significant decline in both Vi IgG and IgA titres between one
and 13 months after Vi-TT vaccination. In addition, we show
that individuals primed with Vi-TT and boosted with Vi-PS at
19-23 months, have an increase in Vi antibody titre, reaching
similar levels as those measured one month post-prime. The
response to the Vi-PS boost is characterised by a significant
increase in circulating Vi-specific IgA ASCs, but no rise in IgM
or IgG ASCs nor Vi-specific memory B cells.
Significant waning of both anti-Vi IgG and IgA from
measured peak levels was observed sooner in the Vi-TT group
than amongst those primed with Vi-PS, perhaps as a
consequence of the high titres induced with Vi-TT vaccination.
While Vi-TT generated higher anti-Vi IgG and IgA responses
one month after vaccination, at 13 months post-vaccination,
anti-Vi IgG levels were comparable between Vi-TT and Vi-PS
recipients while anti-Vi IgA titres were comparable at four
months for both vaccine groups and remaining so at 13
months. Thirteen months after primary vaccination, anti-Vi
IgG and IgA titres remained elevated above baseline (both Vi-
TT and Vi-PS), and with the elevation above baseline retained at
20 months in the small cohort of Vi-TT recipients (n=5) sampled
at this late time point. These data suggest that Vi-TT induces Vi
antibody responses that persistent for up to at least 20 months,
comparable to the Vi-PS vaccine. Although persistence of higher
antibody levels does not necessarily translate to direct protection
from disease, recent analysis of Vi-specific serological correlates
of protection against typhoid underline the importance of anti-
Vi IgG antibody in preventing disease after exposure as well as
highlighting a lesser described significance of anti-Vi IgA
(24,26).
The rise in antibody titre one month post Vi-TT prime was
similar to that seen in response to the Vi-rEPA and Vi-CRM
conjugate vaccines (15,16,27,28). At 19 –23 months (median
interval of 20 months) following Vi-TT vaccination the GMT of
anti-Vi IgG was 65.7 EU/ml (n= 5), similar to the GMT of 82.2
FIGURE 4 | Vi specific IgG and IgA antibody response to Vi-TT conjugate prime and Vi-PS booster vaccination. Vi antibody titres were measured with ELISA, and
comparison of titres between time points was performed. Interval between D28 post-prime and PV Booster ranged from 19 to 23 months. Adjusted p-values are
indicated. **p ≤0.01. PV, pre-vaccination.
FIGURE 5 | Vi specific IgG and IgA pre-boost antibody titre versus fold change between pre-boost and D28 post-boost anti-Vi IgG and IgA antibody titre. Vi
antibody titres were assayed with ELISA.
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EU/ml reported by Mohan et al. using the same assay at 720 days
(23.7 months) post-Vi-TT vaccination in an Indian population
(17). Multiple episodes of natural exposure or subclinical
infection in the Indian population both prior to and following
vaccination might explain the slightly higher titre observed
compared with our cohort of UK adults. We have seen no
significant boosting of titres after single exposure to S. Typhi
in the context of our challenge model, the higher level of
persisting antibodies in endemic populations might be due to
various factors such as exposure to other strains with higher Vi
expression, multiple exposures, or other differences in the host
or environment.
Demonstrating medium-term maintenance of immunological
features which correlate with protection against typhoid, our data
contribute to the mounting evidence supporting the use of Vi
conjugate vaccines. Subsequently, this Vi-TT vaccine has been
administered to over 10,000 children aged between 9 months and
16 years in Nepal, a typhoid endemic country, where it has
demonstrated an efficacy of 81.6% after 1 year (20). Pakistan was
the first country to introduce Vi-TT into its routine immunization
programme and here more than 10 million doses have been
administered to combat the outbreak of extensively drug resistant
(XDR) typhoid in the Sindh province (21). While Vi antibody titre
data can be used to guide expectations of duration of protection
afforded by vaccination, there is currently no definitive protective
threshold. It is important to look at other immune parameters such
as antibody effector functions and cellular responses to understand
the mechanism and duration ofprotectionaffordedbyVi
vaccination, in the most comprehensive analysis of features which
protect following Vi-TT to date, measures of IgA quantity and
binding were found to be the most strongly associated with
protection (24).
FIGURE 6 | Vi-PS booster vaccination induces an anti-Vi IgA ASC response but no change in anti-Vi IgG or IgM ASCs. Peripheral ASC numbers were determined
by ex vivo ELISpot, and comparison of titres between time points was performed *p ≤0.05; **p ≤0.01. PV, pre-vaccination. Prime data collected from group A
participants who were also enrolled in group C, n = 5. Boost data collected from all group C participants, n = 10.
FIGURE 7 | Vi-PS booster vaccination does not induce a significant increase in circulating IgG or IgA Vi-specific memory cells. Activated memory cells were assayed
by memory ELISpot, and comparison between vaccine arms was performed. Adjusted p-values are indicated. PV, pre-vaccination.
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Conjugate vaccines hold promise for improving protection in
view of their increased immunogenicity in infants and because
they are T-dependent antigens. Furthermore, greater initial B-
cell responses have been shown to be associated with greater
antibody persistence at one year of age suggesting that the
magnitude of initial germinal centre reaction is an important
long-term determinant of protection (29).
Of note, the majority of participants in this study were exposed
to S. Typhi one month after vaccination (‘challenge’). Comparison
of longitudinal antibody profiles from these individuals with a
small set of vaccinated but unchallenged participants suggests
exposure to S. Typhi in this context does not significantly boost Vi
antibody titres. The effect of repeated exposures, more similar to
real-life settings where Vi seropositivity is related to levels of
typhoid transmission is unknown (30).
The Vi-PS booster in previously Vi-TT-vaccinated volunteers
induced a significant increase in antibody titre within 1 month of
vaccination (both IgG and IgA). Analysis of changes at the level of
the individual indicate the magnitude of the increase in antibody
levels was inversely associated with circulating antibody level at the
time of the booster. This relationship might reflect a negative
feedback mechanism, potentially in place to prevent aberrant
responses. This is consistent with clinical trials of a diphtheria-
conjugated Vi typhoid vaccine, which showed that an additional
conjugate vaccine booster 6 weeks after an initial vaccination did
not increase antibody titres above those observed post-prime, and
another in which revaccination again had no incremental effect on
Vi antibody titre in relation to post-prime (27,31). In contrast a
study in which two to five year olds received Vi-rEPA noted a ten-
fold rise in Vi IgG in response to revaccination four weeks after the
primary dose (15). The differences noted in the response to a
second dose of a conjugate vaccine may relate to differences in the
products, including the amount of unconjugated polysaccharide in
the vaccine and the carrier protein. Repeated doses of Vi-PS
vaccines; may induce hyporesponsiveness, but several studies
demonstrate that a second dose of Vi-PS given at appropriate
intervals gives rise to antibody levels at least as high as those
following the prime, and the WHO recommend revaccination
every 3 years (17,32,33). Here we show that boosting with a Vi-
polysaccharide after a Vi-conjugate prime can successfully return
Vi IgG levels to those present after the primary vaccine, but no
evidence for an enhanced response to the second dose. These
findings may imply that natural exposure in endemic settings could
also boost immunity after administration of conjugate vaccines and
potentially sustain protection in populations. Boosting of antibody
levels may not be enough to directly confer protection, since
antibody dependent effector functions have been shown to more
closely correlate with protection rather than titre alone, as
demonstrated by Jin et al (24). Assessment of antibody effector
functions after boosting was beyond the scope of this project.
Previous studies have shown that polysaccharide-conjugate
vaccines induce long lived antigen specific memory B cells (34).
We have previously shown that Vi-TT vaccination induces Vi
specific memory B cells which are significantly elevated above
baseline 28 days after vaccination, while Vi-PS showed no
significant change in memory B cell formation (25). Our data
add to this and suggest that Vi specific memory B cells are not
detectable in peripheral blood 19-23 months after Vi-TT
vaccination. Increases in Vi antibodies following booster
vaccination with Vi-PS are not accompanied by an expansion
in Vi specific memory B cells detectable in peripheral blood,
while boosting with Vi-TT was not explored within this study.
Administration of a pneumococcal polysaccharide vaccine boost
following a conjugate vaccine prime was observed to give rise to a
decrease in capsular polysaccharide-specific memory B cells, in
keeping with the hypothesis that polysaccharide antigens drive
pre-existing switched memory B cells into terminal
differentiation and are unable to replenish the pool of memory
B cells (35). While one possibility for the failure to detect
memory B cells in response to the Vi-PS boost is that the
circulating Vi specific memory B cells are low frequency or
that the activated memory B cells may home to bone marrow and
are therefore they are not detected in the peripheral blood. It is
also possible that the Vi-PS boost may negatively impact Vi-
specific memory B cells, and hence potentially antibody
responses to subsequent Vi exposure. We were unable to
compare boosting with Vi-PS with Vi-TT as this was not
studied - the project was specifically designed to generate Vi
specific ASC for the production of monoclonal antibodies after a
Vi-PS boost. Boosting with Vi-TT would also induce expansion
of tetanus toxoid ASCs making it less suitable for Vi specific ASC
isolation. However, exploring boosting with Vi-TT to restore Vi
antibody levels and memory B cells should be considered; studies
of memory B cell responses to pneumococcal conjugate vaccines
set a strong precedent for this (36,37).
Limitations of our work include the relatively short period of
study; later time points are much needed to inform on longer
term persistence of antibodies; and the small number of
individuals who received a Vi-PS booster vaccination, the
original aim of revaccination being to stimulate responses for a
study to generate Vi monoclonal antibodies. Data from phase III
trials of Vi-TT over a longer period are expected imminently and
will address the first of these shortcomings.
In conclusion, we present data demonstrating that typhoid
conjugate vaccination gives rise to initially higher Vi antibody
titres compared with Vi-PS vaccination, with antibodies persisting
at similar levels to those in response to Vi-PS at the end of the first
year of vaccination. This study provides further evidence to support
the use of conjugate vaccines in endemic areas, to both reduce the
burden of typhoid fever and to combat growing antibiotic
resistance. Future studies should explore the merits of different
strategies to boost Vi responses following Vi conjugate vaccination.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by The South Central Oxford A Ethics Committee
Bentley et al. Vi Antibody Persistence and Boost
Frontiers in Tropical Diseases | www.frontiersin.org September 2021 | Volume 2 | Article 7097458

(14/SC/1427). The patients/participants providedtheirwritten
informed consent to participate in this study.
AUTHOR CONTRIBUTIONS
AP, JH, CJ, and EJ conceived and designed the work. CJ, JH, EJ,
MM, JG, and JP carried out the clinical trial. EJ, TB, and CJ
acquired data for the work. EJ, TB, and JH analysed the work. TB,
EJ, JH, CJ, and AP interpreted data for the work. TB, EJ, JH, CJ,
and AP wrote the manuscript. All authors contributed to the
article and approved the submitted version.
FUNDING
This study was funded by The Bill & Melinda Gates Foundation
(OPP1084259) and the European Commission FP7 grant
“Advanced Immunization Technologies”(ADITEC).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fitd.2021.
709745/full#supplementary-material
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Author Disclaimer: The views expressed in this article do not necessarily
represent the views of the UK Department of Health and Social Care, Joint
Committee on Vaccination and Immunisation, or World Health Organization.
Conflict of Interest: AP is Chair of the UK Department of Health and Social
Care’s Joint Committee on Vaccination and Immunisation; and is a member of the
World Health Organization’s Strategic Advisory Group of Experts. The University
of Oxford has entered into a partnership with AstraZeneca on coronavirus vaccine
development.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
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endorsed by the publisher.
Copyright © 2021 Bentley, Jones, Jin, Moore, Gardner, Hill and Pollard. This is an
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provided the original author(s) and the copyright owner(s) are credited and that the
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practice. No use, distribution or reproduction is permitted which does not comply with
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