Persistence of Protection Conferred by Shingrix Against Herpes Zoster
Phase: PHASE4
Conditions: Herpes Zoster
Sponsor: University of Colorado, Denver

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COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD
CAMPUS BOX F-490    TELEPHONE:  303-724-1055   Fax:  303-724-0990 
 
 
Project Title:  Persistence Of Protection Conferred By Shingrix Against Herpes Zoster 
In Older Adults 
NCT04169009 
COMIRB approval date: 09MAR2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol #:  18-0580 
Project Title:  Persistence Of Protection Conferred By Shingrix Against Herpes Zoster      
                        In Older Adults 
Principal Investigator:  Adriana Weinberg, MD; Myron Levin, MD 
Version Date:  March 8, 2022 
 
 
I. Hypotheses and Specific Aims 
Hypotheses  
1) Shingrix (RZV) recipients will have a lower rate of varicella-zoster virus (VZV) 
DNAemia after a live intradermal (ID) VZV challenge (vOka vaccine) than Zostavax 
(ZVL) recipients because of higher and/or more rapid VZV-specific T cell 
responses. 
2) After a live VZV ID challenge, RZV recipients will have the same rate of VZV 
DNAemia whether the challenge was performed <1 or >5 years after vaccination; 
whereas ZVL recipients will have lower rates of VZV DNAemia if they are 
challenged <1 year after vaccinations compared with >5 years.in t 
3) The peak memory VZV and/or gE Th1 responses to RZV mediate protective 
immune responses conferred by RZV against VZV DNAemia after VZV ID 
challenge; early responses may also play a role 
 
Specific Aims/Study Objectives 
Primary Objective 1. To compare the rate of vOka DNAemia within 7 days after ID 
vOka administration in adults who received RZV or ZVL >5y before the challenge. 
 
Primary Objective 2. To compare the rate of vOka DNAemia after ID vOka challenge at 
<1y after RZV or ZVL vaccination and at >5y after vaccination. 
  
Secondary Objective 3. To identify the characteristics of the immune responses to RZV 
that predict the immunologic control of vOka DNAemia after ID vOka challenge at <1y 
and at >5y after vaccination. 
Note: This will include studies to further define epitope-specific immune 
responses, and will measure transcript profile and epigenetic changes in immune cells 
following vaccination. 
 
 
II. Background and Significance 
Background - Herpes zoster (HZ) is a frequently severe infection caused by varicella-
zoster virus (VZV). 60% 
age, although immune compromise is another risk factor for HZ (1,2). Common to these 
two risk factors is a significant decline, to a critical level, of VZV T-cell mediated 

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immunity (VZV-CMI) initially stimulated by the primary VZV infection (childhood varicella)
(3-5). Varicella also results in the lifelong persistence of VZV in sensory ganglia. 
Sporadic reactivation of VZV occurs, but such events typically remain subclinical 
because of VZV-CMI. However, when an adequate VZV-CMI response to reactivation is 
absent, propagation of VZV in the ganglion continues and spreads down the sensory 
nerve to cause the characteristic signs and pain of HZ (6). A live attenuated HZ vaccine 
(ZVL) has been available since 2006 to prevent this, but its efficacy for preventing HZ is 
51% in the 1st 3y after immunizations and drops sharply after 5y, and efficacy decreases 
with the age of the vaccinee (7. Shingrix (RZV), a recombinant VZV glycoprotein E (gE) 
adjuvanted vaccine, which became available late in 2018, is remarkably superior, with 
97% overall efficacy against HZ. RZV protection declines little over time (8). The efficacy 
of RZV is unique compared with any approved vaccines for older adults and any 
candidate vaccines for herpesviruses. The magnitude and persistence of RZV 
immunogenicity represent a paradigm shift in vaccinology that warrants granular studies. 
 
Significance - Central to understanding the superiority of RZV is determining the timing, 
magnitude, and nature of the systemic and local immune events at the site of VZV 
reactivation and replication. Since the milieu of sensory ganglia cannot be directly 
evaluated, this proposal will use intradermal (ID) administration of live attenuated VZV 
vaccine (vOka strain) to simulate reactivated VZV replication in the skin. 
 
Ageing is associated with increasing frailty and multiple concomitant medical conditions, 
which are compounded by an increasing incidence of infections as a consequence of 
immune senescence. The resulting societal burden is evident from increased 
hospitalization and medical costs among older people (9). This is clearly demonstrated 
by HZ, where the incidence, severity, hospitalization, and complications increase 
dramatically after the 6th decade of life (10). This problem will intensify in the future, 
since there are currently 110 million people in the US over 50y of age and there will be 
11). Understanding how a vaccine for older people 
successfully limits a common infection (namely HZ) in ageing individuals may be 
applicable to the general problem of improving the immunogenicity of other vaccines for 
older people. Specifically, this proposal determines how a vaccine shapes the early 
immune response to a live attenuated vOka VZV challenge and limits VZV infection at 
the challenge site. Local intracellular vOka antigen production and DNAemia resulting 
from the challenge provide endpoints for this new model to understand the effect of early 
post-reactivation immune responses on VZV replication.  
III. Preliminary Studies/Progress Report 
The following preliminary studies have been completed in support of this proposal: 
1. We submitted the results of a 1 year study (so far in year 4 of a 5 year study) to 
Nature Vaccine that determined that RZV generates superior VZV-specific T cell 
memory responses compared to ZVL. All of the immunologic assays needed for the 
proposed trial have been validated and appear in our published reports (12). 

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2. We published methods and findings that demonstrated that VZV DNAemia after a
vOka skin challenge correlates with the differentiation and magnitude of VZV-CMI 
responses (13). 
3.  We published on the methods, safety, and immunogenicity of ID vOka (14).  4. 
Experiments in preparation for this proposal that have been completed include: 
a. Measures of plasma biomarkers after different modes of administration of ZVL 
and RZV. 
b. Measures of selected T cell and antigen-presenting cell responses after ZVL and 
RZV. 
c. Discovery of CD4+ and CD8+ epitopes that will be used for tetramer synthesis 
for the advanced immunologic assessments proposed. 
d. Skin specimens have been studied at the Human Immune Monitoring Shared 
Resource at the Anschutz campus using the 7-color Vectra 3 instrument. This 
methodology will be used to study local responses to the ID vOka challenge.  We 
were able to detect VZV and immune cell phenotypes on these skin biopsies.  
 
 
IV. Research Methods 
A. Outcome Measures/Study Endpoints 
Primary Endpoint - Rate of VZV DNAemia after ID vOka challenge. 
Secondary Endpoint – Duration and magnitude of VZV DNAemia and rate of VZV 
RNAemia.  VZV- and gE-specific T cell and other immune cell responses in blood and at 
the site of ID vOka challenge; cytokines levels in blood and at the site of ID vOka 
inoculation; 
transcriptomics 
of 
immune 
cells 
in 
blood 
and 
skin 
biopsies; 
immunohistochemistry of skin biopsies of the ID vOka challenge.  
Exploratory Endpoint –After RZV: Tetramer analysis of the T cell responses; T cell 
epigenome.  
B. Description of Population to be Enrolled 
Inclusion Criteria 
1. Subject able to understand and provide informed consent. 
2. 
0 years  
3. Ambulatory and in generally good health except for common morbidities of older 
people; 
4. Women of non-childbearing potential meet at least 1 of the following: 
a. Achieved postmenopausal status, defined as cessation of regular menses 
for at least 12 consecutive months 
b. Have undergone hysterectomy and/or bilateral oophorectomy 
5. Part A, Cohorts 1 and 2 – Documented evidence of immunization with ZVL 
(Cohort 1; n=35) or RZV (Cohort 2; n=35) at least 5 years previously;  

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6.
Part B, Cohorts 3 and 4 - HZ vaccine naïve - Cohort 3 (n=35) will be vaccinated 
with ZVL at study enrollment, or Cohort 4 (n=35) will be vaccinated with RZV at 
study enrollment. 
 
Exclusion Criteria
1. Inability or unwillingness of a participant to give written informed consent or to 
comply with study protocol. 
2. Prior history of HZ;  
3. Known history of hypersensitivity to ZVL or vaccine components in all study 
candidates and to hypersensitivity to RZV or vaccine components in Cohort 4. 
4. Known neomycin contact allergy or other ZVL components such as gelatin. 
5. Untreated anemia in Cohorts 1 and 2. 
6. Blood products received in the 3 months prior to study enrollment or planned for 
the subsequent week for Part A; Part B requires the same exclusion but extends 
it to the week after the vOka challenge, which is 6 months after completing ZVL 
or RZV administration.   
7. Any confirmed or suspected immunosuppressive or immunodeficient condition 
resulting from disease (e.g., malignancy, HIV), or immunosuppressive/cytotoxic 
therapy. 
8. Use, or anticipated use, of immunosuppressants or other immune-modifying 
drugs during the period starting 30 days prior to ID ZVL challenge through 7 days 
after.  This includes chronic administration of corticosteroids (>14 consecutive 
days of predn
–acting 
immune-modifying agents, or immunosuppressive/cytotoxic therapy (e.g., 
medications used during cancer chemotherapy or to treat autoimmune 
disorders). Topical, ophthalmic, intra-articular, or inhaled/nebulized steroids are 
allowed. 
9. Concomitant vaccine received within 2 (inactive) or 4 (live) weeks prior to 
the study for subjects in all Cohorts, and during the first week of the 
study. For subjects in Cohorts 3 and 4, no concomitant vaccine received 
within 2 (inactive) or 4 (live) weeks prior to ID vOka and for the following 
7 days.
10. Women of child-bearing potential. 
11. Pregnancy or breast-feeding. 
12. Participation in a concurrent clinical study in which the subject will be exposed to 
an investigational product (drug or vaccine) during the period starting 7 days 
before the first dose of study vaccine through the completion of the study. 
13. Antivirals with activity against VZV, including acyclovir, valacyclovir, ganciclovir, 
valganciclovir, foscarnet, cidofovir, brincidofovir and penciclovir within 7 days of 
the ID ZVL challenge.  
14. Past or current medical problems or findings from physical examination or 
laboratory testing that are not listed above, which, in the opinion of the 
investigator, may pose additional risks from participation in the study, may 
interfere with the participant’s ability to comply with study requirements or that 
may impact the quality or interpretation of the data obtained from the study. 

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C. Study Design and Research Methods 
Part A; Cohorts 1 and 2 
Seventy subjects with documented evidence of a HZ vaccine at least 5 years previously, 
divided equally between recipients of ZVL (Cohort 1) or RZV (Cohort 2) - 35 in each 
group - will be enrolled.  Participation will consist of 4 visits over the course of 7-10 days 
in which blood is drawn, a challenge dose of vOka is given, and a skin biopsy is 
obtained. 
 
ID vOka challenge live attenuated VZV (as 1 dose of licensed ZVL; Zostavax) will be 
prepared by mixing 0.35 ml of accompanying diluent with the vaccine and injecting 2 
side-by-side intradermal doses of 0.125ml each, for a total of 0.25 ml (about ¾ of the 
normal dosage or 13,857 plaque forming units) intradermally in the non-dominant deltoid 
region, using a NanoPass MicronJet600 device, on D0. Blood (60 ml) will be drawn prior 
to vaccination (D0), and at Days 1, 3, and 7. A skin biopsy (4 mm punch biopsy) will be 
done at the injection site, on the first 20 subjects (up to 25, if replacements needed) in 
each vaccine group; five each of these 20 will be biopsied on Day 0, 1, 3, or 7 in the 
order in which the participants enter the study. The Day 0 biopsy will be done on the 
contralateral arm in the deltoid region prior to vaccination.  Initially every subject enrolled 
will be required to do the biopsy; this is reflected in the consent.  The consent form will 
be amended to remove references to the biopsy procedure once the number of subjects 
needed for the biopsies is reached. 
 
Subjects in the ZVL group will be offered RZV vaccine (to comply with ACIP 
recommendations for shingles immunization), to be administered beginning  60 days 
after Day 0.  If subject wishes to receive the RZV vaccine, 2 additional visits will be 
required at Day 60 and Day 120, increasing the duration of participation up to 4 months.  
Subjects will not be reimbursed for these visits but will receive the vaccine free of 
charge. 
 
Schedule of Events – Cohorts 1 & 2 
Cohorts 1 and 2
ZVL/RZV > 5y previously 
Visit 1
Visit 2 Visit 3
Visit 4
Phone
Visit 5
Visit 6 
Day 0
Day 1
Day 3
Day 7 
(+3) 
+ 48 hour 
after V4 
Day 60
(+30) 
Day 120 
(+30) 
Informed consent, review of 
eligibility criteria
 
 
 
 
Medical history and current
medications
 
 
3
3
Blood draw (60ml) 
 
 
 
 
Pre-vaccination body 
temperature
 
 
3 
3 
Vaccination (ID vOka challenge)
 
 
 
 
Punch biopsy of ID challenge 
site1 
1,2 ,or
1 ,or
1 ,or
1
 
Vaccination – Dose 1 RZV3 
 
 
 
3 
Vaccination – Dose 2 RZV3 
 
 
 
 
3 
AE assessment 
 
 
 

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140 subjects (20 in each vaccine group) with 5 each at one of the time points identified 
2Contralateral site only
  3 If prior ZVL
Recruitment – There are very few people in the world who had RZV administered 5 
years previously.  These subjects will come from an experiment done here (COMIRB 
#13-3192) in which we compared immune responses to RZV and ZVL. The 4y post-
vaccination samples are being collected this year, and the 5y sampling will be completed 
between Nov 2019 and Mar 2020. When these older vaccinees complete 5y after 
immunization, they will constitute a unique population eligible for our proposal. To secure 
participation for this proposal, we will contact participants in our current trial (who had 
consented to be contacted about future clinical trials). 
 
Part B; Cohorts 3 and 4 – No Previous HZ vaccine
Part B measures VZV DNAemia and systemic VZV-specific immune responses after ID 
vOka challenge in individuals who received HZ vaccines 6 months prior to the injection.   
 
Cohort 3 (n=35) subjects will be vaccinated with ZVL at enrollment; blood will be 
obtained at Days 0, 1, 3, 7 and 30 days after. Five - six months after receiving ZVL 
subject will be challenged with vOka administration (as described in Part A) followed by 
visits 1, 3, and 7 days later at which blood is drawn. Total of 9 to 11 visits. 
 
These subjects will also be offered RZV vaccine (to comply with ACIP recommendations 
for shingles immunization) to be administered  60 days after vOka challenge. If subject 
wishes to receive the RZV vaccine, 2 additional visits will be required  2 beginning 2 
months after Visit 6, increasing the duration of participation up to 10 months. Subjects 
will not be reimbursed for these visits but will receive the vaccine free of charge. 
 
Schedule of Events – Cohort 3 
Cohort 3 – 
 No Previous HZ Vaccine 
(to be admin ZVL)
Visit 1 Visit 2 Visit 3 Visit 4 Phone Visit 5
Visit 6
Visit 7 Visit 8 Visit 9
Phone
Visit 10
Visit 11 
Day 
0 
Day
1 
Day
3 
Day
7 
Day 
9 
(+3)
Day 
30 
(+7)
Day 
150 
(+44)
 Day 
 151 
Day 
153 
(+2)
Day
157 
(+10)
+48 
hours 
after V9
60 Days
after V6
(+30)
60 days 
after V10
(+30)
Informed consent, review of 
eligibility criteria 
 
 
 
 
 
 
 
 
 
 
Medical history and current
medications 
 
 
 
 
 
 
 
 
Blood draw (60ml)
 
 
 
 
 
 
 
 
 
 
Pre-vaccination body 
temperature
 
 
 
 
 
 
 
 
 
 
Vaccination – Zostavax
 
 
 
 
 
 
 
 
 
Vaccination (ID vOka 
challenge) 
 
 
 
 
 
 
 
 
 
AE assessment
 
 
 
 
 
 
 
 
 
Vaccination – Dose 1 RZV1
 
 
 
 
 
 
 
 
1
Vaccination – Dose 2 RZV1
 
 
 
 
 
 
 
 
 
1
1If subject wishes to receive RZV 

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Cohort 4 (n=35) subjects will be enrolled, have blood (60ml) drawn, and will be 
administered RZV (2 doses –D0, D60) prepared and administered in accordance with 
the package insert, in the clinic.  Participation will consist of 10 visits over the course of 
approximately 9 months.  Blood will be drawn at Day 0, Days 61, 63, 67, and 90.  These 
subjects will receive the challenge dose of vOka 6 months later at Day 240, with blood 
drawn, then drawn again at Days 241, 243, 247.  Participation will consist of 10 visits 
over the course of approximately 8.5 months. 
 
Schedule of Events – Cohort 4 
Cohort 4 –
 No Previous HZ Vaccine 
(to be admin RZV) 
Visit 1 Phone Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8 Visit 9 Visit 10 Phone
Day
0
Day 
7  
(+3)
Day
60
(+14)
Day
61
Day 
63 
Day
67
(+3)
Day
90
(+7)
Day 
240
(+14)
Day
241
Day 
243
Day
247
(+3)
+48
hours
after V 
5, 6, 10
Informed consent, review of 
eligibility criteria
 
 
 
 
Medical history and current
medications 
 
 
Blood draw (60ml)
 
 
 
 
Pre-vaccination body 
temperature 
 
 
 
 
Vaccination – Dose 1 RZV
 
 
 
 
 
Vaccination – Dose 2 RZV
 
 
 
 
 
Vaccination (ID vOka 
challenge)
 
 
 
 
 
AE assessment 
 
 
 
 
 
D. Description, Risks, and Justification of Procedures and Data Collection Tools 
Risks 
Zostavax- The risks are those associated with a licensed vaccine for people 50 years 
and older.  Subjects will be given the relevant Vaccine Information Sheet (VIS).
ID vOka challenge (as 1 dose of licensed ZVL; Zostavax) –The intradermal route was 
previously studied in ~200 subjects without a noteworthy safety signal (13). The device 
for ID administration is licensed and was used in a prior study (COMIRB #11-1210).  
Some subjects will be receiving ZVL for the second time.  There is no recommendation 
for a second dose of ZVL, but we completed a study (COMIRB #10-1461) in which 200 
participants safely received a second dose of ZVL.  Furthermore, ZVL is recommended 
after prior herpes zoster, which is analogous to re-exposure (as with our ID challenge) 
to the virus of herpes zoster.  Very rarely, a brief, painless blistery rash limited to the 
site of injection may develop. 
Shingrix – This vaccine will be administered according to the package insert. The risks 
are those associated with a licensed vaccine for people 50 years and older.  Subjects 
will be given the Vaccine Information Sheet (VIS).   

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As with all vaccines or drugs, rarely an allergic reaction could occur – a rash, hives or
even difficulty breathing. 
 
Risk of receiving multiple shingles vaccines – two doses of RZV have been safely 
administered to prior ZVL recipients, and also to individuals who had prior HZ. This was 
done prior to licensure and mentioned in the ACIP recommendation, indicating no 
concern with an aberrant immune response in this setting.  
Blood draws – 60 ml of blood will be taken at each blood draw for a total of 240 ml in 1 
week for subjects in Cohorts 1, 2, 3 and 4.  Subjects in Cohort 3, receiving ZVL, will 
have a total of 540 ml drawn over the course of the study.  Subjects in Cohort 4, 
receiving RZV, will have a total of 600 ml drawn, over the course of the study. Neither 
will have more than 240 ml drawn in a week.  The risks of phlebotomy are minimal and 
include pain, swelling, bruising, or infection around the vein, although this is very 
uncommon.   
Punch biopsy – The skin biopsy will be taken at the ID vOka challenge site (except for 
Day 0 participants, in which case it will be done on the contralateral arm).  The area will 
be numbed; then a punch biopsy device will be pressed into the skin removing a small 
circle of skin.   
Risks include local pain, potential for infection, and scarring at punch site, and possible 
allergic reaction to the numbing medicine. This procedure is being overseen by a 
dermatologist skilled in this procedure. This procedure is important because it will 
indicate what the local immune response to the herpes zoster virus is in people who 
previously received either ZVL or RZV. 
 
Genetic testing – In order to do epitope mapping and to create tetramers we will do HLA 
typing of white blood cells. 
Benefits 
There is no known benefit from this study, but this research may influence future 
decisions made by participants concerning booster doses of HZ vaccine.  It may also led 
to improved vaccines for older persons. ID ZVL may further enhance protection. This is 
likely, but is unstudied.  Some participants may receive RZV, which is recommended to 
prevent herpes zoster. 
 
E. Safety Reporting 
For this study, adverse events (AE) will be collected for 48 hour after each study 
procedure and for 7 days after vOka administration. Additionally, AEs will be collected 
for 7 days after administration of ZVL to Cohort 3, and RZV to Cohort 4 participants.  
Participant will be asked at each visit:  “Have there been any significant changes to your 
health since we saw you last?  New medications?  Hospitalizations?”  
 

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An adverse event will include any untoward or unfavourable medical occurrence 
associated with: 
Study intervention:  ID vOKA – AEs starting within 7 days after injection and 
 ZVL – Subjects in 
Cohort 3 receiving ZVL on Day 0 will be followed for AEs for 7 days.  RZV – 
Subjects in Cohort 4 receiving RZV on Day 0 and Day 60 will also be followed for 
AEs for 7 days after each injection.  
until resolution. 
 
Study mandated procedures:  blood draw, skin biopsies and injections – Any 
 injection, skin 
biopsy or venipuncture will be recorded and followed until resolution.  (This 
excludes optional RZV vaccination for participants in Cohorts 1 and 3.) 
 
Adverse events will be graded on a scale from 1 to 5 according to the following: 
Grade 1 = mild adverse event.  
Grade 2 = moderate adverse event.  
         Grade 3 = severe and undesirable adverse event. 
Grade 4 = life-threatening or disabling adverse event. 
Grade 5 = death. 
The PI will review AEs on a continuous basis during the study. Both Zostavax and 
Shingrix are FDA-approved vaccines that have been extensively studied.  While no 
unusual vaccine-related SAEs are anticipated, we propose the following halting rules for 
risk management.  The PI will responsible for responding to vaccine-related serious 
adverse events.  Reporting will be to the manufacturers of each vaccine, the IRB, and to 
the NIH Medical Monitor (as required).   
 
       
Attribution of Events 
 
Code
Descriptor
Relationship (to primary investigational product 
and/or other concurrent mandated study therapy or 
study procedure)
UNRELATED CATEGORY
1
Not 
Related
The adverse event is clearly not related:  there is 
insufficient evidence to suggest a causal relationship.
 RELATED CATEGORIES 
2
Possibly 
Related 
The adverse event has a reasonable possibility to be 
related; there is evidence to suggest a causal 
relationship.
3
Related
The adverse event is clearly related. 
Any SAE considered possibly, probably, or definitely related will result in halting of the 
study while an analysis of the event is undertaken.  Any halting of the study and the 
circumstances involved will be reported to the NIH Medical Monitor within 24 hours.  An 
Independent Safety Monitor (ISM), who is an Infectious Disease physician not 

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associated with the study, will review any SAE considered possibly, probably, or 
definitely related, prior to discussion with the research team. When sufficient information 
is available, the PI, the ISM, and the NIH Medical Monitor, will determine if and when the 
study can proceed with additional subjects and under what conditions.  
Death or serious injury caused by the Nanopass device and device malfunctions will be 
reported to the manufacturer as per memo of understanding between the manufacturer 
and the FDA.
Any subjects who experiences an SAE considered related after the first dose of Shingrix
vaccine will not be given a second dose.
F. Potential Scientific Problems 
1.
Recruitment will take considerable effort.  Potential challenges include the 
invasive procedures (punch biopsy) involved, and finding persons in the 
community who’ve never received HZ vaccination.  
2.
DNAemia may not be absolutely equivalent to viremia.  
3. Early events in skin may not mimic those in ganglionic environment. 
4. vOka challenge may not be similar to natural infection with VZV.  Immune 
response to each may be different.
G. Data Analysis Plan
An independent bio-statistician will review data prior to submission for publication.
For analysis of kinetics of DNAemia: 
For sample size estimation, we assumed an incidence of viremia of 86% in the ZVL 
-fold reduction in 
the 
RZV
group. 
The 
2-fold 
reduction 
is 
a 
conservative 
estimate, considering that the 
efficacy is 2.9-fold higher 5y after 
RZV vs. ZVL. With 19 participants per group, we have 80% power to detect a 2-fold 
difference in viremia with 
=0.05. However, a larger sample size is needed to achieve 
the 2ary objectives. 35 participants per group will allow us to have sufficient power in 
correlation analyses of our primary outcome measure with 2 measures of the immune 
responses, which may represent clusters of several analytes that group together using 
PCA or other dimensionality reduction method. For the primary objective, logistic 
regression models will be fit using VZV DNAemia defined in Section 13.2. as a 
dependent variable and vaccine group as the primary independent variable and adjusted 
for potential confounders such as age at vaccination, age at ID vOKA challenge, sex, 
and/or race as appropriate. For all regression models fit to the data, 95% confidence 
intervals will be reported based on Wald statistics in conjunction with appropriate 
asymptotic normal distributional theory. Secondary analyses will include the duration of 
DNAemia defined as the last day after ID vOKA administration when the VZV DNA PCR 

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was positive, the magnitude of VZV DNAemia, defined as the peak viral load, and the 
-RNA PCR positive result during the 
first week after the ID vOKA challenge. 
For analysis of soluble factors and phenotypic markers:
We will generate descriptive tables of response and compare soluble factors and 
phenotypic markers between the 2 groups using multivariate analysis methods. We will 
use Hotelling's T2 test for the flow cytometric and soluble factors. We will adjust the 
outcome measures for potential confounders and redo the T2 test. If necessary, 
outcome measures will be log-transformed in order to better satisfy the assumptions of 
the T2 method. Cross sectional pairwise comparisons of gene expression will be made 
at 0, 1, 3 and 7d between groups using the DESeq2 package. Genes differentially 
expressed at day 0 will be filtered out at subsequent time points to avoid confounders 
unrelated to the intervention. Differentially expressed genes of interest at 1, 3 or 7d will 
be validated by RT-qPCR and used as input for downstream gene ontology pathway 
analysis. We will use the lists from each time point to investigate trends in gene 
expression over time (e.g. Is gene A upregulated at all time points?). Due to the large 
number of tests, False discovery rate (FDR) will be controlled at the level of 0.05. 
For analysis of soluble markers and relationship to transcriptomic results:
We will generate descriptive tables of vOka replication and immune responses from all 
parameters by vaccine groups. We will use generalized linear models (GLM) to identify 
correlates between local vOka replication and viremia and between local and systemic 
APC, NK and Tconv, soluble markers and gene expression. Correlations will be 
examined between biopsies and systemic data collected at the same, preceding, and 
following time points. We will adjust for age and gender as appropriate and use FDR p-
values.   
For analysis of systemic and local immune responses that limit viremia: 
With the different biological measurements, the major analytical goal will be to determine 
biomarkers that serve as intermediates in the pathway between virus challenge with 
DNAemia.   With all the matched measurements (transcriptomic, soluble marker, VZV-
CMI T cell responses) on the same subjects, we will use mediation analysis methods 
(15) in order to evaluate the mediation potential of these markers.   As a primary 
analysis, we will create a composite mediation score based on the following linear 
combination: 
, where 
denotes an association between molecule m 
with the exposure, and 
is the measurement on molecule m for subject i, i=1,…,n.  
Upon standardizing V, we then can test for mediation using the approach in Vittinghoff et 
al. (16). Based on the available n = 70, we performed power analyses based on the 
Vittinghoff et al. approach to evaluate power for mediation of the composite marker.  We 
assumed that the probability of outcome is 56% based on our preliminary data and that 
we are testing at a significance level of 0.05. Using the powerMediation package in R 
(https://cran.r-project.org/web/packages/powerMediation/index.html), 
with 
the 
given 
sample size, we will have at least 80% power of identifying mediating effects of V that 

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are at least 1.96 on the log odds ratio scale based on the proposed extended Sobel test 
given in Vittinghoff et al. (16).   
 
Sample size: Sample size is 70. Calculation for predictability of this sample size is 
described above.
H. Summarize Knowledge to be Gained 
This project is designed to determine why RZV is a superior vaccine compared to ZVL, 
and to understand why it is uniquely effective in protecting elderly people. This will be 
accomplished by understanding the timing, magnitude, and nature of the systemic and 
local immune events at the site of VZV reactivation and replication. Since the milieu of 
sensory ganglia cannot be directly evaluated, this proposal will create a model for this 
purpose, which will be intradermal administration of live attenuated VZV vaccine (vOka) 
strain to simulate reactivated VZV replication in the skin. Both systemic and local 
responses to this challenge will be determined. Understanding how a vaccine for older 
people successfully limits a common infection (namely HZ) in ageing individuals may be 
applicable to the general problem of improving the immunogenicity of other vaccines for 
older people.  
I. Future Studies 
Serum plasma and white blood cells will be archived for future studies of immune 
responses to VZV. 
 
J. References 
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