PLOS One | https://doi.org/10.1371/journal.pone.0314828 June 26, 2025 1 / 13

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Citation: Tchos KF, Ridzon R, Haidara MC,
Dabitao D, Akpa E, Camara D, et al. (2025)
Investigating immune amnesia after measles
virus infection in two West African countries:
A study protocol. PLoS One 20(6): e0314828.
https://doi.org/10.1371/journal.pone.0314828

Editor: Javier Antonio Benavides-Montaño,
Universidad Nacional de Colombia Campus
Palmira, COLOMBIA

Received: November 22, 2024

Accepted: April 24, 2025

Published: June 26, 2025

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STUDY PROTOCOL

Investigating immune amnesia after measles
virus infection in two West African countries:
A study protocol

Karine Fouth Tchos 1, Renée Ridzon 1, Mory Cherif Haidara2, Djeneba Dabitao3,
Esther Akpa1, Daouda Camara2, David Vallée4, Mariam Coulibaly3, Sekou Camara2,
Jamila Aboulhab1, Mahamadou Diakité 3, Bassirou Diarra3, Samba Diarra3, Ilo Dicko3,
Alyson Francis 5, Cécé Francis Kolié2, Michel Koropogui2, Caeul Lim4, Seydou Samaké3,
Sally Hunsberger1*, Moussa Sidibé2, Ray Y. Chen 1, Issa Konate3, Seydou Doumbia3,
Abdoul Habib Beavogui2, Kathryn Shaw-Saliba 1

1 Division of Clinical Research, NIAID/NIH, Rockville, Maryland, United States of America, 2 Centre
National de Formation et de Recherche en Santé Rurale (CNFRSR) de Maferinyah, Conkary, Guinea,
3 University Clinical Research Center, University of Sciences, Techniques and Technologies of Bamako,
Bamako, Mali, 4 Clinical Monitoring Research Program Directorate (CMRPD), Frederick National
Laboratory for Cancer Research, Frederick, Maryland, United States of America, 5 Systex, Inc, Rockville,
Maryland, United States of America

* sally.hunsberger@nih.gov

Abstract

“Investigation of Immune Amnesia Following Measles Infection in Select African

Regions” (ClinicalTrials.gov Identifier: NCT06153979) is a prospective, observa-

tional, longitudinal study being conducted in two West African countries; Guinea

and Mali. Acute measles virus (MeV) infection has been shown to result in a loss

of pre-existing immunity (immune amnesia). MeV-induced immune amnesia has

not been studied in West Africa where continual MeV outbreaks occur. Additionally

previous studies have relied on naturally occurring exposures to viruses to exam-

ine the immune systems ability to create antibodies. Thus, the overall goal of this

protocol is to investigate the impact of MeV infection on pre-existing immunity to

endemic pathogens in West Africa, observe the effect of a subsequent exposure to a

novel pathogen (rabies vaccine), and measure the frequency of subsequent health-

care visits. A total of 256 children aged 1–15 years are being enrolled into one of

two study arms: those with acute MeV infection (cases) as confirmed by laboratory

testing and without (controls). Controls must be immune to MeV (have IgG). Blood

samples are collected at multiple time points including screening (Day 0), at an

optional visit to repeat IgM serology for inconclusive or negative Day 0 results (Day

7–10), and during follow-up visits on Day 14, Week 13, and Week 52. These blood

samples will be tested to evaluate both humoral and cellular immune responses to

a panel of viruses, bacteria, and parasites, including pathogens endemic to West

Africa. To explore how recent MeV infection may affect the child’s ability to respond

to a new exposure, all participants will receive a rabies vaccine (as a controlled

PLOS One | https://doi.org/10.1371/journal.pone.0314828 June 26, 2025 2 / 13

stimulus) at one of two timepoints post Day 0 visit. Biological samples will be col-

lected after vaccination to assess if the rabies vaccine response differs: 1) between

cases and controls, and 2) based on the time since acute MeV infection. In addition,

the study team will collect information on healthcare encounters during the year-long

follow-up to determine if there is a difference in the number of encounters by study

group. The findings of this study will further the understanding of the MeV immune

amnesia phenotype by understanding its impact on endemic pathogens and subse-

quent immune response following infection.

Introduction

Measles virus (MeV) infection is a major cause of death globally for children under
five years of age [1–3]. There are reports of increases in cases in many countries,
including in West Africa [4,5]. The Expanded Program on Immunization in Guinea
and Mali calls for two doses of measles vaccine starting at 9 months [6,7]. According
to WHO/UNICEF national immunization coverage estimates for the African region in
2022, 69% of children received the first measles vaccine and 45% received the sec-
ond [6]. The rates in Mali were very close to these averages, at 70% for the first dose
and 44% for the second dose, but Guinea lagged farther behind, at 47% and 3%,
respectively [8]. These rates are far below the 92–95% level required for herd immu-
nity and control of community transmission of measles [9]. This is primarily because,
over the past decade, West Africa has experienced measles outbreaks following
large-scale public health emergencies, such as the Ebola epidemic and the COVID-
19 pandemic, which have disrupted global routine childhood vaccine coverage
[10–12]. Armed conflict has also disrupted routine vaccinations in many countries,
resulting in a disproportionate number of cases of vaccine preventable diseases,
including measles [13,14]. Finally, some vaccination campaigns may have used
vaccines where appropriate storage conditions were not maintained due to a lack of
available resources or equipment. Thus, the presence of global measles outbreaks
underscores the importance of studies that will contribute to a better understanding of
the effect of MeV infection on morbidity and mortality.

MeV infection not only directly causes morbidity but is also associated with
increased susceptibility to other infections and/or increased hospitalizations [15–19].
Measles vaccination has been associated with larger reductions in morbidity and
mortality than would be expected if caused by acute MeV infection alone [19–24].
While there is a strong immune response to MeV infection that confers life-long
immunity to MeV there is some evidence of a loss of existing immunity to other
pathogens. This phenomenon, known as “immune amnesia” [25], is associated with a
loss of memory B cells and reduced antibody repertoires from previous infections and
vaccinations [26,27]. MeV-induced immune amnesia has been characterized in ani-
mal studies with ferrets and rhesus macaques and has also been examined in Euro-
pean populations [26,28,29]. Given the current outbreaks of measles in West Africa,
this study aims to determine whether measles-induced immune amnesia occurs in

purpose. The work is made available under
the Creative Commons CC0 public domain
dedication.

Data availability statement: This article does
not report any data and the data availability
policy is not applicable.

Funding: The author(s) received no specific
funding for this work.

Competing interests: The authors have
declared that no competing interests exist.

PLOS One | https://doi.org/10.1371/journal.pone.0314828 June 26, 2025 3 / 13

African regions and affects immune responses to endemic pathogens such as Mycobacteria tuberculosis, Plasmodium
species, and arthropod-borne viruses (e.g., yellow fever virus or dengue). The hypothesis of this study is that acute MeV
infection induces a loss of pre-existing immunity to pathogens prevalent in select African regions. Compared to those who
do not have acute MeV infection, those with acute MeV infection will: 1) lose antibody diversity from previous infections
and/or vaccinations compared to baseline; 2) have an altered immune response to a known exposure at different time-
points (early vs. late) post measles infection (exposure timing is controlled by using the rabies vaccine as a controlled
immune stimulus) 3) have an increased incidence of illnesses or healthcare encounters in the year following acute MeV
infection.

The data gathered in this study will better characterize the effect and mechanism of measles-induced immune amnesia
in children. The design of this study is described.

Materials and methods

Ethical statement

National Institute of Allergy and Infectious Diseases (NIAID) is the sponsor of the study. The main protocol and each
of the site-specific appendices received approval from local ethics committees and/or health authorities prior to study
implementation including the University of Sciences, Techniques and Technologies Bamako Mali #2023/210/CE/USTTB
-Sept 18, 2023, and Guinea CNERS # 145/CNERS/23 -Sept 8, 2023. Prior to initiation of any study procedures, all study
participants had an informed consent signed by a parent or guardian, and an assent as applicable, in a language they
understand.

Clinical study design

Study Overview: “Immune Amnesia Following Measles Infection in Select African Regions” (ClinicalTrials.gov Identifier:
NCT06153979), is a prospective, observational, longitudinal study being conducted in West Africa to investigate the
effects of MeV infection on pre-existing immunity, vaccine response, and susceptibility to subsequent illnesses. A total
of 256 children are being enrolled into 1 of 2 arms: acute MeV infection (cases) or no acute MeV infection (controls).
The sample size calculations are based on the primary endpoints: geometric mean RVNA titers and mean MIPSA
score for the global antibody analysis and are detailed in the Statistical Aspects section. Briefly, there is 90% power to
detect a standardized difference of 0.85 between cases and controls for the geometric mean RVNA titer for the groups
that receive the vaccine early vs. late. There is 90% power to detect a standardized difference in the MIPSA score of
1 between cases and controls. Both of the standardized differences (0.85 and 1.00) are as large as those observed
previously [26]. Acute measles is defined as signs and symptoms consistent with measles and confirmed in the labora-
tory using measles-specific RT-PCR on upper respiratory specimens and anti-measles IgM on blood samples. Controls
are defined as an absence of signs and symptoms, negative RT-PCR, negative on anti-measles IgM, and positive on
anti-measles IgG. (Controls who are otherwise eligible at screening but with a negative anti-measles IgG are vacci-
nated and enrolled.) Measles RNA (YouSeq RT-PCR measles, Cat no. YSL-qP-EC-Measles-100), anti-measles IgM
(Abcam ELISA, Cat no. ab108751), and anti-measles IgG (Abcam ELISA, Cat no. ab108750) will be tested using the
indicated commercially available kits that are verified in each laboratory. Blood samples are collected at: screening
(Day 0), at an optional visit to repeat IgM serology (Day 7–10), and during follow-up visits on Day 14, Week 13, and
Week 52. Plasma and, where available, PBMCs will be cryopreserved. Minimum blood volumes are collected in accor-
dance with local IRBs. The blood samples will be used to address the primary, secondary, and exploratory objectives
as outlined in Table 1. For children positive for measles, the upper respiratory sample and/or plasma from screening
visit (Day 0) will be used to identify the measles genotype. In the children who test negative for measles but present
with signs and symptoms consistent with measles, we will explore other viral pathogens as outlined in the exploratory
objectives in Table 1.

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Table 1. Study objectives and endpoints.

Objectives Endpoints Justification For Endpoints

Primary

To determine if MeV infection induces a
loss of pre-existing immunity (immune
amnesia) to endemic pathogens at Week
13 after baseline in children in select African
regions.

Mean change in a panel of antibody levels over 13 weeks
as measured by multiplex serological methods (e.g.,
Molecular Indexing of Proteins by Self-Assembly (MIPSA)
with an updated VirScan library and additional libraries
with endemic bacteria and parasite) and targeted ELISAs
for confirmation using plasma collected at Screening (Day
0) and Week 13.

These methods detect antibodies against
multiple diverse pathogens and epitopes and
assess both neutralizing and non-neutralizing
antibodies.

To determine the effect of MeV infection on
immune response to a controlled immune
stimulus (rabies vaccination) at early and
late timepoints post-infection.

Geometric mean RVNA titer 5–6 weeks after the first
rabies vaccine dose using plasma from D14 (randomiza-
tion) as pre-vaccine and at either Week 13 (early random-
ization) or Week 52 (late randomization) as post-vaccine.

These tests will measure the immune
response and functionality of antibodies
raised against the rabies vaccine.

Proportion of subjects with an RVNA titer ≥ lower limit of
quantification 5–6 weeks after the first rabies vaccine dose
using plasma from D14 (randomization) as pre-vaccine
and at either Week 13 (early randomization) or Week 52
(late randomization) as post-vaccine.

Proportion of subjects with rabies virus neutralizing anti-
bodies (RVNA) titer ≥ 0.5 International Units per milliliter
(IU/mL) as measured by rapid fluorescent focus inhibition
test 14 days after the last PrEP regimen vaccination using
plasma from D14 (randomization) as pre-vaccine and at
either Week 13 (early randomization) or Week 52 (late
randomization) as post-vaccine.

Secondary

To determine if there is an increase in
healthcare system encounters in the year
following enrollment in children with recent
MeV infection compared to those without
recent MeV infection.

Mean number of non-study sick visit healthcare system
encounters during the 1-year follow-up.

Visits to the healthcare system for sick visits
in the year following enrollment in children
with recent MeV infection will help determine
the clinical impact of the immune amnesia in
an objective manner.

To determine if MeV infection induces a
loss of pre-existing immunity (immune
amnesia) to endemic pathogens at Week
52 after baseline in children in select African
regions.

Mean change in a panel of antibody levels over 52 weeks
as measured by multiplex serological methods (e.g.,
MIPSA with an updated VirScan library and additional
libraries with endemic bacteria and parasite) and targeted
ELISAs for confirmation using plasma from screening (Day
0) and Week 52.

These methods detect antibodies against
multiple diverse pathogens and epitopes.
These represent the spectrum of both neutral-
izing and non-neutralizing antibodies.

Exploratory

To assess the appearance and continued
production of B and T cells, especially ASCs
from peripheral blood mononuclear cells
(PBMCs) in circulation following MeV infec-
tion in children 1–15 years old in a subset
of participants, depending on availability of
collected blood samples.

Mean change in cell number and functionality from base-
line (screening) to 14 days. PBMCs from screening (Day
0) and D14.

Assessing the number and functionality of the
B and T cells including ASCs will give insight
into the impact of MeV on B cell development
and humoral immunity.

To attempt to identify the etiology of illness
in children who present with signs and
symptoms consistent with measles and are
screened but not enrolled due to negative
measles PCR and IgM.

Unbiased next generation sequencing for pathogen dis-
covery directly from the oropharyngeal (OP)/nasopharyn-
geal (NP) swab and/or plasma from screening (Day 0).

This may identify other pathogens that may
mimic the clinical presentation of MeV in chil-
dren who have symptoms but test negative.

To identify the genotypes of MeV collected
in the study.

Next generation sequencing or targeted Sanger sequenc-
ing directly from the OP/NP and/or plasma from screening
(Day 0).

This will allow identification of different
genotypes of MeV in the countries enrolling
participants.

(Continued)

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To explore how recent MeV infection affects the ability to respond to a new previously unencountered pathogen
at two different timepoints following MeV infection, participants will receive rabies vaccine pre-exposure prophylaxis
(PrEP) with Verorab inactivated rabies vaccine (Sanofi) as part of the study. PrEP is given as none of the children
in the study would have previously been vaccinated or exposed to rabies. PrEP is intended to protect the recipient
in case they are subsequently exposed to rabies. Previous studies have shown that children who receive a vaccine
following MeV infection may have a lower antibody response to the vaccine [30]. The rabies vaccine was chosen
because it is an immunogen to which most participants have not been exposed and thus can be used to measure
the immune response to a newly encountered antigenic stimulus. Additionally, as rabies is estimated to cause 59,000
human deaths annually worldwide with the highest mortality in Africa [31,32], the provision of rabies PrEP is a benefit
to participants in the study [33]. All children in each arm will receive rabies vaccination (3-dose series at Days 1, 7 and
28 given as PrEP), with the first dose randomized to either Week 8 or Week 47 after enrollment. WHO now recom-
mends a 2-dose PrEP schedule [32] but we decided to use the older 3-dose schedule from the Verorab package insert
(https://www.medicines.org.uk/emc/files/pil.15572.pdf) to give the acute measles cohort children the best chance of
developing an immune response in the event of persisting immune amnesia. Biological samples will be collected after
vaccination to assess if the immune stimulus (rabies vaccine) response differs: 1) between children with and without
MeV infection, and 2) based on the timing of the receipt of the rabies vaccine. A participant who is potentially exposed
to rabies during study participation will be provided rabies post exposure prophylaxis (PEP) with Verorab vaccine, with
or without human rabies immune globulin, based on published recommendations [33]. The study schedule is depicted
in Fig 1.

The study team will collect, to the extent possible, information on healthcare encounters that take place during the year-
long follow-up. This will be compared between cases and controls to determine if there is a difference in the number of
encounters by study group.

The study objectives and endpoints are shown in Table 1.

Eligibility

The study inclusion and exclusion criteria are shown in Table 2. Children aged 1–15 years who are eligible after initial
screening are enrolled in Group 1 of children with acute MeV infection (cases) or Group 2 of children without acute MeV
infection (controls) eligible for the study and will be referred for care. An upper respiratory specimen (oropharyngeal (OP)/
nasopharyngeal (NP)) for MeV PCR and plasma for measles IgM/IgG serology are collected. Plasma and PBMCs are also
collected at screening for research assays. PBMCs will only be collected in Mali due to operational constraints and lack of
dry ice in Guinea. Anyone suspected of having measles at the time of screening will be treated with Vitamin A 200,000 IU
by mouth each day for 2 days, as recommended by the WHO [34].

Objectives Endpoints Justification For Endpoints

To characterize the phenotype of the rash
and/or Koplik spots in children who present
with signs and symptoms consistent with
measles who test positive for measles (poly-
merase chain reaction [PCR] and/or IgM
positive) and who test negative for measles
(PCR and IgM negative).

Differences in rash patterns/presentation in children who
test positive versus those who test negative for measles
based on analysis of photographs.

This will allow for a comparison of rash
and/or Koplik spot presentation in poten-
tial cases that end up testing positive or
negative for measles in the laboratory. This
will complement the exploratory objective
of identifying the etiology via unbiased next
general sequencing in the participants who
test negative for measles. In addition, we
can contribute images of measles rash on
dark skinned children and Koplik spots to the
medical literature.

https://doi.org/10.1371/journal.pone.0314828.t001

Table 1. (Continued)

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Study visits according to initial protocol

A schematic depiction of the study visit timing is shown in Fig 2. During Visit 0 on Day 0, demographic, medical history,
vaccination history and concomitant medication information are collected, and a physical exam is performed. Mid upper
arm circumference is obtained in children ≤59 months. Due to the turnaround time for the MeV PCR and measles IgM/IgG

Fig 1. Study schedule. Abbreviations: AE, adverse event; HIV, human immunodeficiency virus; Ig, immunoglobulin; MUAC, mid-upper arm circum-
ference; NP, nasopharyngeal; OP, oropharyngeal; PCR, polymerase chain reaction. (X) = Each participant will receive only one 3-dose series of rabies
vaccine according to randomization to early group (vaccination at Visits 2, 3, and 4) or late group (vaccination at Visits 8, 9, and 10). a EARLY rabies vac-
cination: Only participants randomized to EARLY rabies vaccine attend Visits 2, 3, 4. Visit 2 occurs 8 weeks after Day 0; Visit 3 occurs 7 days after Visit
2; Visit 4 occurs 21 to 28 days after Visit 2 AND at least 14 days after Visit 3. b LATE rabies vaccination: Only participants randomized to LATE rabies
vaccine attend Visits 8, 9, 10. Visit 8 occurs 47 weeks after Day 0; Visit 9 occurs 7 days after Visit 8; Visit 10 occurs 21 to 28 days after Visit 8 AND at
least 14 days after Visit 9. c Day 0 is defined as the day screening occurs and the first research blood sample is collected. Inclusion/exclusion criteria
evaluation for enrollment will be completed when PCR and measles IgG/IgM and HIV results are available, generally by Day 14. All potential participants
(or guardian/parent) will be informed of eligibility status by a designated study staff by phone, if possible, prior to the Day 14 scheduled study visit or at
the Day 14 scheduled study visit. Ineligible participants may be invited to the site to discuss test results and referral for measles vaccination if serum IgG
and IgM are both negative. d Vital signs include temperature, heart rate, and respiratory rate; may be obtained prior to rabies vaccine administration, if
clinically indicated. e MUAC in children 12 to 59 months old. f HIV testing for children younger than 24 months will be performed via PCR and repeated
with Rapid Diagnostic Test (RDT) at end of study to adhere to national guidelines; HIV RDT will be performed at screening for participants 24 months
and older and will not be repeated at end of study. g HIV and measles serum IgM/IgG results must be reviewed for eligibility confirmation prior to Day
14. h Blood will be collected for hemoglobin test via finger and/or heel stick. Hemoglobin and urine pregnancy rapid test results must be reviewed prior
to venous blood sample collection. i Females of child-bearing potential must have a negative pregnancy test prior to enrollment and each rabies vaccine
administration. j Measles vaccination to be administered to control participants (through the study or referral to Ministry of Health vaccination program) if
serum measles IgG and IgM are both negative. k 5 mL is the maximum volume that will be obtained at each blood draw. l Vitamin A will be administered
at Day 0 to all participants with clinical measles per WHO guidelines: 2 oral doses of 200,000 IU given 24 hours apart. The first dose will be administered
at the study site and the second will be provided to the parent to be administered 24 hours later.

https://doi.org/10.1371/journal.pone.0314828.g001

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in the laboratory, eligibility as a case or control is confirmed at Visit 1 on Day 14. Measles vaccination will be administered
to controls who do not have a positive measles IgG at this visit. Once enrolled, participants will be randomized to receive
3 doses of rabies vaccine as PrEP early (doses at Weeks 8, 9, 11–12) or late (doses at Weeks 47, 48, 50–51). At Weeks
26 and 39, a healthcare questionnaire will be administered. Participants’ last visit is scheduled for Week 52. This longer
follow-up period is to allow examination of antibody repertoire dynamics paired with number of healthcare encounters,

Table 2. Study inclusion and exclusion criteria.

Inclusion Criteria Exclusion Criteria

1. Aged 1–15 years.
2. Ability of the participant’s legal or culturally acceptable representative to provide informed consent.
3. Ability to give assent, as appropriate.
4. Stated willingness of parent/guardian and participant as appropriate, to comply with all stufdy

procedures.
5. Willingness to receive rabies vaccine.
6. Meet the criteria for assignment to Group 1 or Group 2, as follows:
a. Group 1, cases (acute MeV infection):
• Clinical signs and symptoms suggestive of acute MeV infection (Koplik spots or skin rash)

AND
• Laboratory confirmed measles:
◦ Upper respiratory specimen (swab) PCR for measles positive.
OR
◦ Serum IgM for measles positive.
b. Group 2, controls (no acute MeV infection):
• No clinical signs and symptoms suggestive of acute MeV infection (Koplik spots or skin rash)

AND
• Upper respiratory specimen (swab) PCR negative for MeV AND
• Serum measles IgM negative AND
• Serum measles IgG positive and previously vaccinated for measles (2nd dose will be offered

if appropriate). If serum measles IgG is negative, participant must be willing to be vaccinated
regardless of prior measles vaccine history to meet this criterion.

1. HIV infection or any other immunosuppres-
sive condition or medications.

2. Pregnant or lactating.
3. History of prior measles or immunologic

evidence of prior measles in the absence of
prior measles vaccination.

4. Severe anemia, defined as hemoglobin less
than 8 g/dL.

5. Any acute or chronic condition which, in
the opinion of the investigator, constitutes a
contraindication to participation in this study.

https://doi.org/10.1371/journal.pone.0314828.t002

Fig 2. Study schematic. 1Research blood samples. 2 Measles vaccination in controls if confirmed to have negative serum measles IgG. 3 Rabies vac-
cine Dose 2 is given 7 days +2 days after Dose 1, and Dose 3 is given at Day 21 to Day 28 after Dose 1 and at least 14 days after Dose 2. 4Healthcare
utilization assessment. Color coding: Blue: visits with research blood samples. Orange: optional visit with repeat for measles serology (IgM) for inconclu-
sive results from the Day 0 test. Purple: visit with measles vaccination in controls if confirmed to have negative measles IgG. Green: All activities and vis-
its pertaining to the rabies vaccine. Black: visits with healthcare utilization assessment. Red: Period in which the healthcare encounters will be recorded.

https://doi.org/10.1371/journal.pone.0314828.g002

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as previous studies have shown that children may remain susceptible to infections for up to 1–3 years following measles
infection. Windows around study visits are relatively short so that the targeted endpoints can be captured accurately.
Visits that occur out of the window periods are permitted under special circumstances, to avoid missed visits. A research
blood sample is collected at Days 0 (screening) and 14(pre-vaccines), and Weeks 13 (up to 6 weeks after first early rabies
vaccine dose) and 52 (up to 6 weeks after first late Rabies vaccine dose, and 1 year follow up visit); prior to collection of
blood, hemoglobin is checked in all participants via a fingerstick, and a urine pregnancy test is obtained in females starting
at their reproductive age. Anyone found to be pregnant is withdrawn from the study. If the hemoglobin is < 8 g/dl at any
follow up blood draw visit, the participant remains in the study, but a blood sample is not collected at that time.

Study sites

This study is being conducted in Guinea and Mali, which were chosen based on measles epidemiology, existing research
infrastructure, and ongoing partnerships between their Ministries of Health, and in Mali, the Ministry of Higher Education,
and the National Institute of Allergy and Infectious Diseases (NIAID) in the United States.

The Partnership for Clinical Research in Guinea (PREGUI) aims to conduct and implement a program of national and
international, high-quality research on public health priorities in Guinea, and to build and develop sustainable research
capacity. This study is implemented at two sites under PREGUI, both located within treatment centers for diseases with
epidemic potential at referral hospitals in Forécariah and Mafèrinyah. These sites receive referrals from surrounding
health centers, health posts and communities and are each served by a multidisciplinary team.

The University Clinical Research Center (UCRC) is located at the University of Sciences, Techniques, and Technolo-
gies of Bamako in Mali. Its mission is to promote clinical research and training at international standards in Mali and within
the West Africa region. The UCRC offers competence in clinical research including clinical operations, laboratory diagno-
sis and exploration of immune function, data management, pharmacy and community engagement. The research team
recruits participants from the Commune VI Reference Health Center and the Bakorobabougou Community Health Center,
both in Bamako.

The first participant was enrolled in Guinea on 01/16/2024 and in Mali on 02/26/2024. The study is currently enrolling
and the expected completion of enrolment is 05/31/2025 for both Mali and Guinea.

Amendment

After screening the first 33 potential participants with suspected measles, laboratory results confirmed measles was pres-
ent in only 15 (45.5%), showing that the clinical diagnosis does not always match the validated laboratory assay results.
Due to concerns that true measles cases were being missed because the IgM test was possibly conducted too early, prior
to development of the IgM response but after the viremic period leading to false negative results [35–38], the protocol
was amended. Thus, children highly suspected of measles based on their clinical presentation but who tested negative
on both PCR and IgM at day 0 may have an optional visit between days 7–10 to retest for measles IgM [35–38]. Adding a
day 7–10 re-test allows more time for the IgM response to develop. Optional photography of participants being screened
as potential cases was also added to the protocol. Photographs of the rash in confirmed measles cases will be used to
train study staff to improve recognition of acute MeV infection in dark skinned children and serve as an educational tool
for the medical community. In screened potential cases who test measles negative, these photos will be coupled with the
information gained in the exploratory aim of unbiased sequencing of other pathogens to determine the phenotype of other
febrile rash illnesses that may otherwise be clinically diagnosed as measles.

Laboratory and stored specimens

The RNA is extracted from the NP/OP swabs collected in viral transport media (VTM) at screening and tested by RT-PCR
for MeV. Remaining VTM samples will be stored for sequencing and genotyping of MeV. Samples from children with signs

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and symptoms consistent with measles but who test negative are stored and can be examined for other viruses using
pathogen discovery sequencing methods. Measles IgG and IgM are measured using an enzyme linked immunoassay on
plasma samples. The screening tests used in each country (hemoglobin, pregnancy, measles serology, HIV) all under-
went verification prior to study start to ensure the reproducibility and accuracy of the results. Plasma collected at baseline
(Visit 0), week 13 (Visit 5) and week 52 (Visit 11) will be tested at a commercial laboratory in the United States to measure
antibody binding to epitopes of select viruses, bacteria and parasites using validated multiplex serological methods (e.g.,
Molecular Indexing of Proteins by Self-Assembly [MIPSA] technology with an updated library and additional libraries with
endemic bacteria and parasites) [26,39]. PBMCs are isolated from blood specimens from Mali, stored in liquid nitrogen,
and will be analyzed for antibody secreting cells (ASC). Plasma obtained following rabies vaccination will be shipped to a
commercial laboratory accredited to perform Rapid Fluorescent Foci Inhibition Test (RFFIT) to ensure neutralizing anti-
body (RVNA) response to the rabies vaccine and to compare cases to controls and those randomized to early vaccination
versus late vaccination. Samples not used for analyses will be stored and managed electronically in the local bioreposito-
ries under appropriate temperature with constant monitoring.

Safety reporting and monitoring

Safety reporting and monitoring is done according to national regulatory guidelines and International Council for Harmon-
isation (ICH). A clinician with appropriate expertise (Principal Investigator [PI]/Designee) will assess adverse event (AE)
severity, according to the “Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events” (https://
rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdf) and determine the seriousness, relatedness and expect-
edness of the event. This may be done in coordination with the sponsor, NIAID. Per study specific standard operating
procedure, only AEs related to study procedures will be followed through resolution or until the local investigator judges
that the event has stabilized, and no additional follow-up is required. Safety events will be tracked and submitted to the
local ethics committees according to each country’s requirements as described in the site-specific appendices. All AEs will
be recorded on source documents.

AEs related to the research procedures and all serious adverse events (SAEs) will be recorded in the research data-
base, except for Grade 1 or 2 AEs that are expected. SAEs and Unanticipated Problems will be reported to Sanofi Pasteur
with coded data according to the terms of a confidential Clinical Trials Agreement.

Statistical aspects

The overall sample size of the study is 256 participants, 128 enrolled as cases and 128 as controls. The sample size
accounts for a 6.5% rate of dropout from the study and participants who drop out will not be replaced. The sample size
is calculated to detect a difference in the RVNA geometric mean titer between the measles cases and controls who were
either vaccinated at the early time point or at the late time point). If each vaccination group (early/late) contains 128 par-
ticipants with 64 cases and 64 controls, there will be 90% power to detect a standardized difference in RVNA geometric
mean titer of 0.85 between cases and controls under each of the vaccination conditions. There will also be 90% power
to detect an interaction between group (measles/controls) and time (early/late) if the effect size is 1.19. The interaction
corresponds to showing a different response between cases and controls at early and late rabies vaccine stimulus times.
There is 90% power to detect standardized mean differences of 1.00 when looking for differences in the MIPSA panel of
antibody responses between cases and controls in each age group. This calculation controls for multiple comparisons.
This effect size was similar to those observed in the paper by Mina et al [26].

Analysis of the rabies antibody response will be performed using multivariate linear regression on the log
10

titer values,
including terms for case/control group, early/late vaccine, country and an interaction between group and timing of vaccine.
Antibody responses to a panel of antigens will be compared between case/control groups using t-tests on change from
baseline. Separate analyses will be performed for the 2 age groups (1–5 and 6–15 years). A Bonferroni correction will be

PLOS One | https://doi.org/10.1371/journal.pone.0314828 June 26, 2025 10 / 13

used to maintain a 2-sided 0.05 error rate by adjusting for the number of antibodies in the panel. A Poisson analysis will
be used to compare the number of encounters with medical providers between groups.

Control participants who become infected with measles or participants from either group who become pregnant at any
time during the study will be censored at the time of infection or pregnancy. A participant who has a potential rabies expo-
sure and receives rabies PEP during the study before s/he is scheduled (randomized) to receive rabies vaccination as
PrEP will be excluded from the primary analysis of the rabies vaccine but included in the immune amnesia analysis. Par-
ticipants who receive at least one dose of rabies PrEP (as randomized) will be analyzed as planned. A Statistical Analysis
Plan (SAP) will be prepared for the study before analyses begin.

Study conduct, data management, operational logistics and challenges

The study leadership consists of a core team of research physicians, statisticians, laboratory research scientists, clinical
research pharmacist and operational experts from the Division of Clinical Research (DCR) of NIAID and both countries.
Both countries have clinical research staff and infrastructure supported by DCR. The study protocol was developed collab-
oratively. The country teams are responsible for submission for ethical review, continuing review reports, quality man-
agement plans, protocol deviations, and SAE reports per country regulations and as requested by NIAID. At all stages,
regular teleconferences and visits are held and include study leadership, country scientific/medical laboratory, data
management and clinical operations teams, and serve as a forum for information exchange on topics such as recruitment,
case adjudication, visit adherence, as well as providing feedback and suggestions on study procedures. A Publication
Policy has been developed to guide the drafting and review of all study-related abstracts, presentations, and manuscripts,
and a Publication Committee that includes representatives from NIAID and each of the country teams will be formed. For
requests for secondary use of biospecimens, the Publication Committee will be approached to approve access to bio-
specimens. Electronic case report forms (CRFs), core standard operating procedure (SOP) documents and manuals of
operation (MOP) were developed along with site-specific documents.

The study was designed to use electronic data capture into the Clinical Data Management System (CDMS). Malian
staff enter data from study-specific paper source documents. Guinean staff use direct data entry into tablets in an online
mode, where the data are saved automatically to the CDMS. If the internet connection is temporarily disrupted, the data
are captured and uploaded to the CDMS once service is restored. This hybrid approach allows for more flexibility across
sites, reduces need for printing CRFs, and requires less transcription of data by the sites. Direct data entry also allows
sites to address any queries that arise from programmed data checks at time of entry. The completed data are then
reviewed by the central data management team for consistency and accuracy. Randomization is also performed electroni-
cally in the CDMS.

Specimen kits with standard barcodes are used for specimen collection. The specimens collected are stored in-country
under proper conditions with constant temperature monitoring and documented in an electronic system.

A monitoring plan is used for central monitoring, to facilitate compliance with GCP, guidelines and regulations, identify
key activities and specify data to be reviewed for the study. Each plan was reviewed by the study teams in Guinea and
Mali and the Measles Study protocol team to ensure adherence to the protocol, laboratory, and GCP requirements. In per-
son and virtual trainings were conducted with the study staff in each country. Refresher trainings are offered as needed.

Each country has encountered challenges with study implementation. The Guinean team is working on enhancing its
ability to use electronic systems for study documents and sample management, manage a heavy workload for the avail-
able laboratory staff, and ensure participants have a good understanding of study processes. The Malian team is working
on increasing the number of screened volunteers by involving more community health centers in referring suspected mea-
sles cases to the team. Also, to enhance use by community mobilizers and to sensitize community members about the
study, flipbooks, a consenting tool depicting the study procedure in easily understood language with images, have been
translated into Bambara, the most spoken language in Mali. Because the sites experience power losses that compromise

PLOS One | https://doi.org/10.1371/journal.pone.0314828 June 26, 2025 11 / 13

refrigeration, all study vaccines are stored off site where a continuous power supply is assured and transported to the
sites as needed. Finally, due to the waxing and waning measles cases in both countries, enrollment is taking longer than
expected.

Conclusion

We have designed and launched an international observational study to investigate immune amnesia following MeV
infection in Guinea and Mali, which have recurrent measles outbreaks. Despite challenges of collaborative protocol devel-
opment, operationalization and need for multiple authorizations to import rabies and measles vaccines, the study was suc-
cessfully initiated in 2024. This effort will increase understanding on how measles immune amnesia affects the response
to a previously unexposed pathogen at different time points post-acute measles infection, characterize MeV genotypes cir-
culating in these regions, and provide insights into other viral causes of maculopapular rash illnesses in children with dark
skin. This study will add to the global understanding of measles from regions previously understudied, and where measles
outbreaks continue to occur due to inconsistent access to vaccines.

Supporting information

S1 File. The Immune Amnesia Following Measles in Select African Regions clinical research protocol version
3.0 11 March 2024.
(DOCX)

S2 File. SPIRIT international standard checklist for the Immune Amnesia Following Measles in Select African
Regions clinical research protocol.
(DOCX)

Acknowledgments

We thank PREGUI study team members, Centre National de Formation et de Recherche en Santé Rurale de Mafèrinyah,
Centres de Santé Amélioré de Mafèrinyah, Hôpital Préfectoral de Forecariah, Community leaders in Maferyniah and Fore-
cariah, Guinea; UCRC study team members, Centre de Santé Communautaire de Bakorobabougou, Centres de Santé
de Reference Commune VI, Direction Générale de la Santé et de l’Hygiène Publique, Direction Régionale de la Santé of
Bamako and Koulikoro, Community partners, Mali; Diane Griffin (deceased) and Jessica Rubens, Johns Hopkins Univer-
sity. We are grateful to Sanofi-Pasteur for contributions.

Author contributions

Conceptualization: Karine Fouth Tchos, Renée Ridzon, Mory Cherif Haidara, Djeneba Dabitao, Esther Akpa, Daouda
Camara, David Vallée, Sekou Camara, Jamila Aboulhab, Mahamadou Diakité, Bassirou Diarra, Ilo Dicko, Alyson
Francis, Cécé Francis Kolié, Sally Hunsberger, Ray Y. Chen, Issa Konate, Seydou Doumbia, Abdoul Habib Beavogui,
Kathryn Shaw-Saliba.

Methodology: Karine Fouth Tchos, Mory Cherif Haidara, Djeneba Dabitao, Esther Akpa, Daouda Camara, Sekou
Camara, Mahamadou Diakité, Bassirou Diarra, Ilo Dicko, Cécé Francis Kolié, Michel Koropogui, Caeul Lim, Seydou
Samaké, Sally Hunsberger, Moussa Sidibé, Ray Y. Chen, Issa Konate, Seydou Doumbia, Abdoul Habib Beavogui,
Kathryn Shaw-Saliba.

Writing – original draft: Karine Fouth Tchos, Renée Ridzon, Kathryn Shaw-Saliba.

Writing – review & editing: Karine Fouth Tchos, Renée Ridzon, Mory Cherif Haidara, Djeneba Dabitao, Esther Akpa,
Daouda Camara, David Vallée, Mariam Coulibaly, Sekou Camara, Jamila Aboulhab, Mahamadou Diakité, Bassirou

PLOS One | https://doi.org/10.1371/journal.pone.0314828 June 26, 2025 12 / 13

Diarra, Samba Diarra, Ilo Dicko, Alyson Francis, Cécé Francis Kolié, Michel Koropogui, Caeul Lim, Seydou Samaké,
Sally Hunsberger, Moussa Sidibé, Ray Y. Chen, Issa Konate, Abdoul Habib Beavogui, Kathryn Shaw-Saliba.

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