Effects of Exposure to Per- and Polyfluoroalkyl Substances (PFAS) on Innate and Adaptive Immune Responses to Tetanus-diphtheria (Td) Vaccination Among Adults in a Community-based Panel Study
Phase: PHASE4
Conditions: Innate Inflammatory Response, Vaccination, Diphtheria, Tetanus, Environmental Exposure, Vaccine, Antibodies, Adult, Immunity, Pollution; Exposure
Sponsor: Rutgers, The State University of New Jersey

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Effects of exposure to per- and polyfluoroalkyl 
substances (PFAS) on innate and adaptive immune 
responses to tetanus-diphtheria (Td) vaccination 
among adults in a community-based panel study 
 
 
 
Document date: June 3, 2025 
 
NCT number: NCT06588530 
 

INTERVENTIONAL
RESEARCH PROTOCOL TEMPLATE
(HRP-503a)
STUDY INFORMATION

Title of Project:
Effects of exposure to per- and polyfluoroalkyl substances (PFAS) on innate and 
adaptive immune responses to tetanus-diphtheria (Td) vaccination among adults in a 
community-based panel study

Principal Investigator Name
Robert Laumbach, MD, MPH, CIH, DABT

Principal Investigator Div. & Dept.
EOHSI/Environmental & Occupational Health
Rutgers School of Public Health

Principal Investigator Contact Info: 
Laumbach@eohsi.rutgers.edu
Rutgers Environmental and Occupational Health Sciences Institute
170 Frelinghuysen Rd, Room 204
Piscataway, NJ 08854
+1-848-445-6084

Protocol Version and Date:
V3: July 11, 2024
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 1 of 14
Table of Contents
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1.0
Research Design
1.1
Purpose/Specific Aims
1.2
Research Significance
1.3
Research Design and Methods
1.4
Preliminary Data
1.5
Sample Size Justification
1.6
Study Variables
1.7
Drugs/Devices/Biologics
1.8
Specimen Collection
1.9
Data Collection
1.10
Timetable/Schedule of Events
2.0
Project Management
2.1
Research Staff and Qualifications
2.2
Research Staff Training
2.3
Other Resources
2.4
Research Sites
3.0
Multi-Center Research
4.0
Subject Considerations
4.1
Subject Selection and Enrollment Considerations
4.2
Obtaining Identifiable Information About Non-Subjects
4.3
Number of Subjects
4.4
Consent Procedures
4.5
Special Consent Populations
4.6
Economic Burden and/or Compensation For Subjects
4.7
Risks of Harm/Potential for Benefits to Subjects
5.0
Special Considerations
5.1
Health Insurance Portability and Accountability Act (HIPAA)
5.2
Family Educational Rights and Privacy Act (FERPA)
5.3
Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations)
5.4
General Data Protection Regulation (GDPR)
5.5
 NJ Access to Medical Research Act (Surrogate Consent) 
6.0
Data Management Plan
6.1
Data Analysis
6.2
Data Security
6.3
Data Safety And Monitoring
6.4
Reporting Results
6.5
Secondary Use of Data
7.0
Research Repositories – Specimens and/or Data
8.0
Approvals/Authorizations
9.0
Bibliography
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 2 of 14
1.0 Research Design
1.1 Purpose/Specific Aims
The purpose of the study is to investigate how exposure to Perfluorononanoic acid (PFNA) modifies 
immune responses to vaccination. We hypothesize that PFNA suppresses the innate immune response 
to vaccine stimulus, consequently leading to a decrease in antibody production. To assess relationships 
between exposure to PFAS and innate and acquired immune responses to a vaccine stimulus, we will 
use a combined approach involving omics technologies and traditional immunological assays. This 
study is a supplement to an existing protocol (“Human health effects of drinking water exposures to 
per- and poly-fluoroalkyl substances (PFAS): A multi-site cross-sectional study”, Pro2020001052). 
A.  Objectives 
1.
Evaluate associations between PFAS levels, markers of innate immune activation, and changes in 
specific antibody levels.
2.
Investigate the relationship between PFNA levels and modifications in immune specific 
transcriptomic and metabolomic responses to vaccination.
B.  Hypotheses / Research Question(s)
We hypothesize that increasing PFNA exposure is associated with a suppressed innate immune, 
measured as pro-inflammatory cytokines and CRP, which leads to a diminished antibody response 
to vaccination. We aim to use transcriptomic and metabolomic pathway analysis to identify cellular 
responses that support this hypothesis.
1.2 Research Significance
Exposure to per- and polyfluoroalkyl substances (PFAS), recognized as "forever" chemicals due to their 
persistent nature, poses a significant threat to public health. With more than 98% of Americans having 
detectable levels of PFAS in their blood, these compounds have been linked to immune suppression by 
observations of increased infectious disease risks and diminished antibody response to vaccination, 
including routine tetanus-diphtheria vaccination in children [1-15]. Previous studies have demonstrated 
differences in the time course of production of specific IgG following tetanus-diphtheria (Td) vaccination in 
a small study of 12 adult participants over 7 time points in a 30-day period [6].  The proposed study will 
focus on exposure to Perfluorononanoic acid (PFNA) in Paulsboro, NJ, and add measurement of innate 
immune responses following the vaccine stimulus. The results of this study will increase knowledge about 
immunotoxicity resulting from PFAS exposure, which has been determined to be the critical effect for human 
health risk assessment by the U.S. EPA (Environmental Protection Agency) and EFSA (the European Food 
Safety Authority) [16,17]. The most recent scientific reviews of immunotoxic PFAS endpoints highlight the 
need for more relevant longitudinal human studies, and probing the immune response through vaccination 
has been a standard approach [18-20]. We will measure several markers of immune response in serum 
and oral fluid samples using standard immunoassays and targeted metabolomic analysis. An innovative 
aspect of our approach is the pilot testing of oral fluid collection as a less burdensome method, compared 
to venipuncture, for serial measurement of immune responses in epidemiological studies. Oral fluid (OF) is 
a biofluid that is a mixture of saliva and non-salivary components, which contains both innate and adaptive 
immune components, including specific immunoglobulins and cytokines [21-30]. OF has been shown to be 
a reliable biomatrix for detecting antibodies that represent serum concentrations, even though antibodies 
in this fluid are often found in lower concentrations when compared to serum. If found to be feasible and 
reliable in this study, this method will be useful in future community-based studies of immune response 
where repeated blood draws can often be a major barrier. Overall, this research project is crucial step in 
developing rational and effective measures to protect public health in the face of widespread PFAS 
exposure.
1.3 Research Design and Methods
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 3 of 14
This study is a pre-post clinical trial. We will assess the impact of PFAS immune responses to routine 
booster vaccination against Tetanus-Diphtheria (Td) over a 30-day period among 20 adults recruited 
from a community in New Jersey that had drinking water contaminated with PFAS, predominantly 
PFNA.  Within the larger study, 775 adults had serum PFAS measurements between July 2021 and 
September 2023.  (“Human health effects of drinking water exposures to per- and poly-fluoroalkyl 
substances (PFAS): A multi-site cross-sectional study”, Pro2020001052). For this study, we will recruit 
participants from the participants in the larger study who agreed to be contacted for future studies.
A.
Research Procedures
All study visits will take place in the local Rutgers study office space (541B Mantua Avenue, 
Paulsboro, NJ) used for the main study. At study visit 1, participants will then have their blood 
drawn (15 mL) by venipuncture by trained research staff. Total amount of blood collected at visit 
one will be 15 mL (1 tablespoon). Participants will self-administer their oral fluid collection using the 
Oracol sampler (https://www.malmed-oracol.co.uk/products/oracol/) by swabbing their gumline for 
1-2 minutes under the supervision of research staff. After baseline sample collection, participants 
will be given a Td booster vaccination. The second, third, and fourth study visits will occur 24, 48, 
and 72 hours after the first visit to capture the innate immune response, while visits five, six, and 
seven will take place 7, 14, and 30 days after the first visit to capture the adaptive immune 
response. At study visits 2-7, participants will have venous blood drawn (15 mL each visit) and will 
collect their oral fluid using the Oracol sampler by swabbing their gumline for 1-2 minutes under 
the supervision of research staff.
Additionally, data collected about the subject collected in the main study (including serum PFAS 
levels, residential history, and history of Td vaccination) will be used in this study.
B.
Data Points
Td specific antibody levels, cytokine levels, CRP, RNA transcripts and metabolites related to 
immune response and activation. 
C.
Study Duration
The study will take approximately one year.  Individual participation will consist of 7 study visits 
over a 30-day period. The initial study visit should take less than 45 mins including the consent 
process and post-immunization observation period, while study visits 2-7 should take 15 minutes. 
Length of participation for each subject will be 30 days. 
D.
Endpoints-NA
1.4 Preliminary Data – NA
1.5 Sample Size Justification
Twenty subjects will be enrolled. Identifying exposures predicting immune and disease outcomes requires 
sufficient power to account for false positives due to multiple comparisons. The goal of 20 adult subjects 
for the vaccination study was selected after statistical power calculations using data from the modeled 
Kielson (2016) study were done. Assuming the Kielson et al. correlation of 0.761 between PFAS level and 
change in diphtheria antibody concentration, then 20 subjects we would have 98.5% power to detect that 
the correlation is different from zero.  Thus, we should have adequate power to detect effect sizes similar 
to those reported by Kielson even with some attrition and participant drop out.  We may see a larger 
correlation as we expect a larger spread in our distribution of PFAS, yielding greater power.  However, even 
with a smaller correlation of 0.7, we would have 95% power.
1.6 Study Variables
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Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
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A.   Independent Variables, Interventions, or Predictor Variables 
Intervention: Participants will be immunized with TENIVAC, which is FDA approved booster vaccine 
against Tetanus and Diphtheria. Td antibody levels will be measured to determine post-vaccination 
antibody status. 
B.  Dependent Variables or Outcome Measures 
Tetanus Toxoid IgG, Diphtheria Toxoid IgG, Cytokines, and CRP concentrations in serum and oral 
fluid. RNA sequencing, and LC/GC-HRMS to identify transcripts and metabolites of immune 
response in serum. 
1.7 Drugs/Devices/Biologics 
A.
Schedule and Administration
Each subject will receive a Td vaccination at the first study visit following consent and distribution 
of vaccine information sheet (VIS). TENIVAC (Tetanus and Diphtheria Toxoids Adsorbed) is an 
active immunization for the prevention of tetanus and diphtheria in persons 7 years of age and 
older. TENIVAC will be administered intramuscularly as recommended in the subject’s deltoid 
muscle at a dose of 0.5 mL which contains 5 Lf of Tetanus Toxoid and 5 Lf of Diphtheria Toxoid. 
Other ingredients per 0.5 mL dose include 1.5 mg of aluminum phosphate (0.33 mg of aluminum) 
as the adjuvant and ≤5.0 mcg of residual formaldehyde. Vaccinations will be administered only by 
medically qualified personnel. 
 
B.
Drug/Device Accountability and Storage Methods
TENIVAC, purchased as either single dose vials or prepared syringes will be stored at the local 
study site in a refrigerator between 2° to 8°C (35° to 46°F) per manufacture recommendation and 
secured behind a locked door only accessible to study staff. Detailed logs for vaccine storage, 
administration, and refrigerator temperature will be kept. Just before use, the single-dose vial or 
syringe will be shaken well until a uniform, white, cloudy suspension results. Preparation, and 
disposal of TENIVAC will be done by trained study staff/research assistants. Syringes and/or vials 
will be disposed of in the proper biohazard sharps container. 
1.8 Specimen Collection
A.
Primary Specimen Collection

Types of Specimens: Blood and oral fluid samples. At each visit, blood samples (15 mL) will 
be collected using standard venipuncture performed by a phlebotomist. Oracol swabs will be 
used to harvest oral fluid rich with GCF (and will be self-collected by the participant under the 
direction and supervision of research staff by gently swabbing their gumline for 2 minutes. In 
total there will be 280 specimens collected for this study (7 blood samples + 7 saliva samples 
per subject x 20 subjects). All specimen collections will happen at the local study site, 541B 
Mantua Avenue, Paulsboro, NJ.

Annotation: The blood and oral fluid samples will be labeled by a study ID and visit number. 
The same study ID will be used for samples/data collected in the main study as in the 
supplemental study.

Transport: The blood and oral fluid samples will be collected at the Paulsboro Study site which 
is also approved by REHS for blood and saliva processing.

Processing: The research assistants will process and aliquot the blood and oral fluid.

Storage: The specimens will be stored in the -80oC freezer at the lab then transported to 
Rutgers EOHSI or shipped to the fee for service analytical lab (Comprehensive Laboratory for 
Untargeted Exposome Science (CLUES), Gangarosa Department of Environmental Health at 
Emory University) by a research assistant with IATA training. 

Disposition: Any excess specimens will be retained by Dr. Laumbach in a -80˚C freezer at 
EOHSI (Room 232). Access to the freezer room is card controlled.  
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 5 of 14
B.
Secondary Specimen Collection - NA
1.9 Data Collection
A.
Primary Data Collection

Location: Subjects will be screened at the local study site in Paulsboro, either in person or 
over the phone. 

Process of Data Collection: Research assistants will do the screening.

Timing and Frequency: Data collection will take place at screening, and at each visit.

Procedures for Audio/Visual Recording: - NA

Study Instruments: A screening questionnaire developed for this study will be used.

Ethnographic Studies, Interviews, Or Observation: - NA

Subject Identifiers: Subject’s contact information (name, email, mailing address, and phone 
number) will be collected to schedule appointments. All documents will be coded by study ID 
and visit number entered directly into the REDCap database. 
B.
Secondary Data Collection-NA
1.10 Timetable/Schedule of Events
2.0 Project Management
2.1 Research Staff and Qualifications
Robert Laumbach MD, MPH, CIH Associate Professor, Environmental and Occupational Health, 
Rutgers School of Public Health. Dr. is a physician-scientist who is board-certified in Family Medicine 
and in Occupational and Environmental Medicine. He conducts clinical and epidemiological research 
on the health effects of environmental contaminants, including air and water pollutants, and has had 
federally funded field projects to study the role of cytokines, including IL-6 and IL-10, in inflammation 
and acute phase responses to exogenous agents, including interactions with psychosocial stress. Dr. 
Laumbach is the Principal Investigator for this study and CDC funded PFAS Multi-Site Study Paulsboro 
study site.  
Pamela Ohman Strickland, PhD, MS Associate Professor of Biostatistics at the Rutgers School of 
Public Health. Dr. Ohman Strickland serves as the Director of the Biostatistics, Epidemiology and 
Research Design Core for the New Jersey Alliance for Clinical and Translational Science (NJ ACTS) 
as well as Faculty Director for the Rutgers University Biostatistics and Epidemiology Services center 
and Director of the Biostatistics Facility Core of the Center for Environmental Exposure and Disease 
(an NIEHS Center) at Rutgers. Dr. Ohman Strickland has collaborated in a wide variety of 
epidemiological studies of acute and chronic effects of air pollution, on biomarkers of inflammation, 
oxidative stress, etc., using methods such as generalized linear models, categorical data analysis, 
factor analysis, path analysis, etc. Dr. Ohman Strickland will serve as a Co-Investigator for this study, 
providing essential epidemiological and statistical support.
Kathy Black, PhD, MPH, Senior Research Associate, Rutgers Environmental and Occupational 
Health Sciences Institute. Dr. Black will act as the study coordinator and will be responsible for the 
training of the study research assistants. Dr. Black has over 20 years of experience coordinating 
environmental health studies. 
Alicia Legard and Adriana De Resende, Clinical Research Coordinators, Rutgers Environmental and 
Occupational Health Sciences Institute. Alicia and Adriana studies have extensive experience in 
managing and performing laboratory procedures, including data analysis, for environmental 
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 6 of 14
contaminant studies and clinical trials. For the current project, they will assist with overseeing the 
clinical trial as needed.
Fatima Haynes, MS, PhD Candidate in Exposure Science at Rutgers School of Graduate Studies.  
Fatima has experience working in molecular toxicology laboratories and on other community health 
research studies. She will serve as a research assistant responsible for participant screening and 
consent, data collection, and analysis. This study will serve as her dissertation project. 
Alanna O’Neil, BS, PhD Candidate in Exposure Science at Rutgers School of Graduate Studies. She 
has a BS in Environmental Science from Juniata College. From July 2021 to present day, Alanna has 
worked at the Paulsboro PFAS Health Study. Among her duties included enrolling participants and 
administering questionnaires. Alanna will provide general research support as needed. 
Tonya Kidd and Nancy Victor will provide general research support and assist with participant screening 
and consent. Both Tonya and Nancy are residents of Gloucester County and also work at the Paulsboro 
PFAS Health Study site.
2.2 Research Staff Training
Only staff working on the main study will also work on this supplement. The staff have been trained 
by Dr. Laumbach in study procedures.
2.3 Other Resources - NA
2.3 Research Sites
All data and specimen collection will take place at the local study office space (541B Mantua Avenue, 
Paulsboro, NJ) including recruitment.
De-identified samples will be analyzed at Rutgers-EOHSI (170 Frelinghuysen Road, Piscataway, NJ 
08854) and at the Comprehensive Laboratory for Untargeted Exposome Science (CLUES), 
Gangarosa Department of Environmental Health at Emory University (1518 Clifton Rd, Atlanta, GA 
30322) for metabolomic analysis on a fee-for service basis.
3.0 Multi-Center Research - NA
4.0 Subject Considerations
4.1 Subject Selection and Enrollment Considerations 
A.
Method to Identify Potential Subjects
Adults who enrolled in the main study who consented to be contacted for future studies will be 
asked to participate in this study. We will recruit participants from the first and fifth quintiles of 
PFNA serum concentrations to maximize contrast in exposure, balanced recruitment from the first 
and fifth quintiles of exposure will be randomized.
B.
Recruitment Details
The study will recruit only main study participants. Previous subjects, who completed their main 
study visit and consented to be contacted for future studies, will be called by a research assistant, 
and asked if they are interested in participating in a supplemental study. If so, an appointment will 
be made for a study visit.
C.
Subject Screening
Subjects are screened for the main study. Adults who participate in the main study will be eligible 
for this supplemental study.
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Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
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
Inclusion Criteria
Adults (18 years of age and older), weigh at least 110 pounds, and who participated in the 
main study may be eligible to participate in this supplemental study.

Exclusion Criteria
o
Pregnancy
o
History of difficult blood draws 
o
History of adverse reaction to prior vaccinations
o
Currently taking immune suppressants
o
Recent dental surgery or dental procedure within 4 weeks of starting study
o
Had a Td booster in the past 10 years
D.
Privacy Protections
Prospective subjects are identified by their participation in the main study. Identifiers for the main 
study are stored in the study REDCap database. Only the study team has access to the 
database.
4.2 Obtaining Identifiable Information About Non-Subjects - NA
4.3 Number of Subjects
A. Total Number of Subjects
20 subjects will be enrolled.
B. Total Number of Subjects If Multicenter Study - NA
C. Feasibility
The main study recruited 775 adults and for the small number of subjects in this study, we expect to be 
able to complete recruiting and testing in a timely manner.
4.4 Consent Procedures
A. Consent Process

Location of Consent Process
Consent will take place in the local study office in Paulsboro. 

Ongoing Consent
Study activities will be discussed with the subject at the start of each visit. All questions will be 
answered.

Individual Roles for Researchers Involved in Consent
Screening and consenting may be done by the coordinator or a research assistant.

Consent Discussion Duration
We expect the consent process to take approximately 10 minutes. The study visit will take 
place immediately after the consent.

Coercion or Undue Influence
Only main study adult volunteers will be asked to participate in the supplemental study. The 
consent form will clarify that participation in the supplemental study is not required.
Subject Understanding
The key study elements will be specifically mentioned during the consent discussion and all 
questions will be answered.

Protecting Privacy
Consent discussions will take place in a private office prior to participation. No additional 
information about the subject will be collected until after consent is obtained.
B.
Waiver or Alteration of Consent Process - NA
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 8 of 14
C.
Documentation of Consent

Documenting Consent
The consent form will be stored in REDCap. Both the participant and study investigator will 
sign the consent form. A copy of the signed consent form will be emailed to the participant.

Waiver of Documentation of Consent (i.e., will not obtain subject’s signature) - NA
4.5 Special Consent Populations - NA
A.
Enrolling Minors-Subjects Who Are Not Yet Adults - NA
B.
Enrolling Wards of the State - NA
C.
Enrolling Non-English-Speaking Subjects - NA
D.
Enrolling Adults Lacking Decision-Making Capacity (Surrogate Consent) - NA
E.
Special Consent Considerations - NA
4.6 Economic Burden and/or Compensation for Subjects
A. Expenses - NA
The subject is not expected to incur any expenses.
B.
Compensation/Incentives
Subjects will be paid $50 for each study visit (up to $350 total).
C Compensation Documentation
Compensation will be documented in REDCap. Subjects will also be asked to sign the subject 
voucher for cash payments.
 
4.7 Risks of Harm/Potential for Benefits to Subjects
A.
Description of Risks of Harm to Subjects

Reasonably Foreseeable Risks of Harm
Td Vaccination: Pain, redness, or swelling where the shot was given, mild fever, headache, 
feeling tired, and nausea, vomiting, diarrhea, or stomachache sometimes happen after Td 
vaccination. People sometimes faint after medical procedures, including vaccination. As with 
any medicine, there is a very remote chance of a vaccine causing a severe allergic reaction, 
other serious injury, or death. 
Venipuncture: Slight pain, some bleeding, or bruising may occur when blood is drawn. A total 
of 105 ml of blood (15 ml per study visit over a 30-day period) will be required for the planned 
assays. 
Oral fluid collection: When swabbing the gumline, there may be minor bleeding if done too 
roughly.  

Risk of Harm from an Intervention on a Subject with an Existing Condition
The study will not enroll subjects with existing conditions related to adverse vaccination 
responses.

Other Foreseeable Risks of Harm
There is a risk of possible loss of confidentiality, but no sensitive information is collected in 
the study. 
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
Observation and Sensitive Information – NA
B.
Procedures which Risk Harm to Embryo, Fetus, and/or Pregnant Subjects -NA
C.
Risks of Harm to Non-Subjects -NA
D.
Assessment of Social Behavior Considerations - NA
E.
Minimizing Risks of Harm
All subjects will be screened prior to scheduling. Subjects with a history of adverse reactions to 
vaccination and difficulty with blood draws are excluded from the study out of an abundance of 
caution. All female subjects will receive a urine pregnancy test prior to vaccination. Subjects will 
be informed in the consent document that they will be given the results of the pregnancy test. 
Although Tetanus-Diphtheria is a routine vaccination, subjects will be provided with a vaccine 
information sheet (VIS) before vaccination to review all information, including risks associated 
with the specific vaccine type. The physician PI will be onsite to administer the vaccine and 
respond to any immediate reactions. Following vaccination, subjects will be visually monitored by 
medical personnel for at least 15 minutes. In case of anaphylactic or acute hypersensitivity 
reactions, Epinephrine hydrochloride solution (1:1,000) or other appropriate agents and 
equipment will be available for immediate use, and 911 will be called. In the case of any adverse 
event, the event will be documented, and a report will be sent to Vaccine Adverse Event 
Reporting System (VAERS) and IRB.

Certificate of Confidentiality - NA

Provisions to Protect the Privacy Interests of Subjects
All data and specimen will be coded by a Study ID. All identifiable information will be stored in 
REDCap and accessible only to the study team.
F.
Potential Direct Benefits to Subjects
Subjects will be boosted against Tetanus and Diphtheria. 
5.0 Special Considerations
5.1 Health Insurance Portability and Accountability Act (HIPAA) - NA
5.2 Family Educational Rights and Privacy Act (FERPA) - NA
5.3 Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations) - NA
5.4 General Data Protection Regulation (GDPR) - NA
5.5 NJ Access to Medical Research Act (Surrogate Consent) - NA
6.0 Data Management Plan
6.1 Data Analysis
Means, standard deviations, percentiles and histograms will summarize the distributions of outcome 
variables. Based on Kielsen (2016), we expect exponential rise in plasma antibody concentrations 
from 4 to 10 days. Thus, in the primary analysis, we will examine the linear increase in log of antibody 
concentrations from 4 to 10 days and whether that increase is modified by exposure to pfas through 
an interaction term between time and PFAS (log-transformed). Integration of exposure, metabolic 
response, and immune response has the potential to provide key insight into mechanisms underlying 
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
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exposure mediated immune suppression following Td vaccine. To examine the assumption that 
there's a linear increase from 4 to 10 days, linear models with cubic splines relative to time since 
immunization will assess goodness of fit across different strata of PFAS exposures. If the time of 
increase varies substantially from the 4 to 10-day assumption, then the initial linear models will be 
adjusted accordingly.
To investigate associations between RNA-seq and metabolomics data with PFAS exposure levels, 
differential analysis will identify genes and metabolites with altered expression/abundance across 
exposure groups. Pathway analysis will assess biological relevance by examining enrichment of gene 
sets and metabolic pathways. Correlation analysis will evaluate the relationship between individual 
gene expression/metabolite levels and PFAS exposure. Longitudinal analysis will assess temporal 
changes in both RNA-seq and metabolomics data following vaccination. Analyses will be adjusted for 
confounders, corrected for multiple testing, and assume stable PFAS concentrations over time. This 
comprehensive approach will elucidate the dynamic relationship between molecular profiles and 
PFAS exposure, advancing our understanding of PFAS toxicity and its impact on human health. For 
analyzing the oral fluid samples, as a feasibility/proof of concept study, no power calculations have 
been done. Accuracy will be assessed by comparison to ELISA reference methods by paired t-test 
and correlation analysis to examine overall bias and reliability. The Bland Altman plot will assess bias 
and homogeneity of error relative to ELISA across the range of values. Mean, standard deviations, 
and coefficients of variation will be calculated to benchmark the method for reference in future 
studies. 
6.2 Data Security
All study data will be stored in REDCap is a secure, password-protected web-based application 
designed to support data capture for research studies. A unique study participant ID will be assigned 
in REDCap at the time of screening. Only study staff will be authorized to have access to the project. 
Only deidentified data will be exported for analysis. De-identification of data will consist of removal of 
all personal identifiers from analytic files. Investigators will keep the data, including links to personal 
identifiers, until study closure.
6.3 Data and Safety Monitoring 
A.
Data/Safety Monitoring Plan
Our plan for data and safety monitoring involves assessing adverse events related to vaccination 
and potential unexpected reactions or interactions. We will continue to monitor and document 
adverse events following vaccination, including both immediate and delayed reactions at study 
visits. Additionally, we will pay close attention to safety parameters specific to the vaccine being 
administered, as outlined in its FDA approval documentation, such as known adverse reactions, 
contraindications, and warnings. Evaluation will occur quarterly throughout the duration of the 
study. The responsibility for data review will primarily lie with the principal investigator, in 
collaboration with a designated safety monitoring team comprising of study staff. The data review 
process will commence at the initiation of the study. Safety information will be collected at each 
study visit. 
B.
Data/Safety Monitoring Board Details
We do not plan to establish a separate data monitoring committee for this study. Instead, safety 
data will be regularly reviewed by the principal investigator and the safety monitoring team. Findings 
from these reviews will be reported to the Institutional Review Board (IRB) and sponsor on a 
quarterly basis. Immediate suspension of the research will be triggered the occurrence of a single 
serious adverse event associated with the vaccine.
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
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6.4 Reporting Results
A.
Individual Subjects’ Results 
No, individual results will not be provided to participants as the research does not involve studying 
anything of clinical relevance.
B.
Aggregate Results
When the study is completed, aggregate results will be posted on the EOHSI website and will be 
presented at community meetings.
C.
Professional Reporting
Results will be presented at professional conferences and published in peer-reviewed journals.
D.
Clinical Trials Registration, Results Reporting and Consent Posting
The study is a clinical trial and will be registered on clinicaltrials.gov. As required, the consent form 
and results will be posted. 
6.5 Secondary Use of the Data- NA
7.0 Research Repositories – Specimens and/or Data
Excess serum and oral fluid specimens may be stored by Dr. Laumbach. The de-identified data collected 
in the study will be linked to the specimens by study ID and will not be distributed to others. The specimens 
will be stored in -80oC freezers at EOHSI Room 232. Freezer temperatures are monitored remotely, and 
notifications are sent in cases of malfunction. Access to the freezer room is controlled by key card. 
8.0 Approvals/Authorizations
IBC approval for saliva and blood processing.
9.0 Bibliography
1.
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in 101 healthy 1-year-old children: associations between levels of perfluorooctanoic acid (PFOA) and 
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02715-4
2.
Grandjean, Philippe et al. “Serum vaccine antibody concentrations in children exposed to 
perfluorinated compounds.” JAMA vol. 307,4 (2012): 391-7. doi:10.1001/jama.2011.2034
3.
Grandjean, Philippe et al. “Serum Vaccine Antibody Concentrations in Adolescents Exposed to 
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Grandjean, Philippe et al. “Estimated exposures to perfluorinated compounds in infancy predict 
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Granum, Berit et al. “Pre-natal exposure to perfluoroalkyl substances may be associated with altered 
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Kielsen, Katrine et al. “Antibody response to booster vaccination with tetanus and diphtheria in adults 
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HRP-503a-TEMPLATE-Interventional Research Protocol 1.11.22
Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 12 of 14
7.
Looker, Claire et al. “Influenza vaccine response in adults exposed to perfluorooctanoate and 
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Pilkerton, Courtney S et al. “Rubella immunity and serum perfluoroalkyl substances: Sex and analytic 
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Shih, Yu-Hsuan et al. “Serum vaccine antibody concentrations in adults exposed to per- and 
polyfluoroalkyl substances: A birth cohort in the Faroe Islands.” Journal of immunotoxicology vol. 18,1 
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FluMist vaccination among healthy adults.” Environmental research vol. 149 (2016): 171-178. 
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Morbidity in a Cohort of Guinea-Bissau Children.” Environmental health perspectives vol. 128,8 
(2020): 87002. doi:10.1289/EHP6517
13. Zell-Baran, Lauren M et al. “Prenatal Exposure to Poly- and Perfluoroalkyl Substances (2009-2014) 
and Vaccine Antibody Titers of Measles, Mumps, Rubella, and Varicella in Children Four to Eight 
Years Old from the Healthy Start Cohort.” Environmental health perspectives vol. 131,12 (2023): 
127018. doi:10.1289/EHP12863
14. Zeng, Xiao-Wen et al. “Prenatal exposure to perfluoroalkyl substances is associated with lower hand, 
foot and mouth disease viruses antibody response in infancy: Findings from the Guangzhou Birth 
Cohort Study.” The Science of the total environment vol. 663 (2019): 60-67. 
doi:10.1016/j.scitotenv.2019.01.325
15. Zeng, Xiao-Wen et al. “Alternatives of perfluoroalkyl acids and hepatitis B virus surface antibody in 
adults: Isomers of C8 Health Project in China.” Environmental pollution (Barking, Essex : 1987) vol. 
259 (2020): 113857. doi:10.1016/j.envpol.2019.113857
16. EFSA Panel on Contaminants in the Food Chain (EFSA CONTAM Panel), et al. "Risk to human 
health related to the presence of perfluoroalkyl substances in food." EFSA Journal 18.9 (2020): 
e06223.
17. US EPA. "Technical Fact Sheet: Drinking Water Health Advisories for Four PFAS (PFOA, PFOS, 
GenX chemicals, and PFBS)." (2022).
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an overview of the current evidence from animal and human studies.” Archives of toxicology vol. 96,8 
(2022): 2261-2285. doi:10.1007/s00204-022-03303-4
19. DeWitt, Jamie C et al. “Exposure to per-fluoroalkyl and polyfluoroalkyl substances leads to 
immunotoxicity: epidemiological and toxicological evidence.” Journal of exposure science & 
environmental epidemiology vol. 29,2 (2019): 148-156. doi:10.1038/s41370-018-0097-y
20. Ehrlich, Veronika et al. “Consideration of pathways for immunotoxicity of per- and polyfluoroalkyl 
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2023, doi:10.1186/s12940-022-00958-5
21. Gaudin, Alexis, et al. "COVID-19 and Oral Fluids." Frontiers in Dental Medicine 1 (2020): 8.
22. Lawrence, Herenia P. "Salivary markers of systemic disease: noninvasive diagnosis of disease and 
monitoring of general health." Journal-Canadian Dental Association 68.3 (2002): 170-175.
23. Lee, Dayong. "Oral Fluid Testing." Principles of Forensic Toxicology (2020): 629-656.
24. Lim, Pei Wen, Johan Garssen, and Elena Sandalova. "Potential use of salivary markers for 
longitudinal monitoring of inflammatory immune responses to vaccination." Mediators of 
inflammation 2016 (2016).
25. Madar, R., S. Straka, and T. Baska. "Detection of antibodies in saliva-an effective auxiliary method in 
surveillance of infectious diseases." Bratislavské Lekárske Listy 103.1 (2002): 38-4
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Protocol Title: PFAS Exposure and Immune Response
Protocol Version: 3
Protocol Version Date: July 11, 2024
PI: Robert Laumbach, MD, MPH, CIH, DABT
Page 13 of 14
26. Panuwet, Parinya, et al. "Salivary bioscience and environmental exposure assessment." Salivary 
Bioscience: Foundations of Interdisciplinary Saliva Research and Applications (2020): 349-370.
27. Randad, Pranay R., et al. "The utility of antibodies in saliva to measure pathogen exposure and 
infection." Salivary Bioscience: Foundations of Interdisciplinary Saliva Research and 
Applications (2020): 287-319.
28. Riis, Jenna L., et al. "Public Health and Industry Applications of Salivary Bioscience." Salivary 
Bioscience: Foundations of Interdisciplinary Saliva Research and Applications (2020): 723-746
29. Riis, Jenna L., et al. "Salivary bioscience, immunity, and inflammation." Salivary bioscience: 
Foundations of interdisciplinary saliva research and applications (2020): 177-213. 
30. Tapia, Milagritos D et al. “Measurement of tetanus antitoxin in oral fluid: a tool to conduct 
serosurveys.” The Pediatric infectious disease journal vol. 25,9 (2006): 819-25. 
doi:10.1097/01.inf.0000232629.72160.bb
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