# Real-world evidence of rabies post-exposure prophylaxis in Serbia: Nation-wide observational study (2017–2019)

Pavle Banović, Dragana Mijatović, Verica Simin, Nenad Vranješ, Eleftherios Meletis, Polychronis Kostoulas, Dasiel Obregon, Alejandro Cabezas-Cruz

► To cite this version:

Pavle Banović, Dragana Mijatović, Verica Simin, Nenad Vranješ, Eleftherios Meletis, et al.. Real-world evidence of rabies post-exposure prophylaxis in Serbia: Nation-wide observational study (2017-2019). Travel Medicine and Infectious Disease, 2024, 58, pp.102697. 10.1016/j.tmaid.2024.102697. hal-04557483

HAL Id: hal-04557483

https://hal.science/hal-04557483

Submitted on 24 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
<figure><img src="image_1.png" /></figure>

# Real-world evidence of rabies post-exposure prophylaxis in Serbia: Nation-wide observational study (2017–2019)

Pavle Banović<sup>a,b,*</sup>, Dragana Mijatović<sup>c</sup>, Verica Simin<sup>d</sup>, Nenad Vranješ<sup>c</sup>, Eleftherios Meletis<sup>e</sup>, Polychronis Kostoulas<sup>e</sup>, Dasiel Obregon<sup>f</sup>, Alejandro Cabezas-Cruz<sup>g</sup>

## ARTICLE INFO

### Keywords:

Rabies

Post-exposure prophylaxis

Essen regimen

Seroconversion

Rabies virus-neutralizing antibodies

## ABSTRACT

**Background:** Rabies remains a deadly zoonotic disease, primarily prevalent in Eastern European countries, with a significant global burden in Asia and Africa. Post-exposure prophylaxis (PEP) is critical to prevent clinical rabies. Serbia, a country with a relatively low animal rabies incidence, has been implementing a 4-dose Essen PEP regimen for 13 years. This real-world study aimed to assess the effectiveness of the 4-dose Essen regimen, considering demographic and clinical factors, after WHO Category III exposure.

**Method:** The study included 601 patients who received the 4-dose Essen PEP and 79 who received an additional 5th dose.

**Results:** Age emerged as a critical factor influencing seroconversion rates after the 4-dose regimen, with older individuals exhibiting lower RVNA titers. Logistic regression indicated a 3.18% decrease in seroconversion odds for each added year of age. The Cox proportional hazards mixed model highlighted age-related risks, with age groups 45–60 and 75–92 at the highest risk of non-seroconversion. Human Rabies Immune Globulin (HRIG) administration was associated with lower RVNA values after the 4-dose regimen, suggesting interference with vaccine immunogenicity among people who received larger doses of HRIG.

**Conclusions:** This study provides valuable real-world evidence for rabies PEP in a non-homogeneous population with potential comorbidities. The results underscore the importance of optimizing PEP strategies, particularly in older individuals, and reconsidering HRIG dosing to improve seroconversion rates.

## 1. Introduction

Rabies is a deadly disease that can be transmitted from animals to humans. It is caused by the rabies virus (RABV) and other related viruses from the *Lyssavirus* genus, Rhabdoviridae family [1]. In Europe, animal rabies is mainly found in Poland [2], Belarus [3], Ukraine [4,5], Russia, Moldova [6] and Romania [7], with sporadic cases reported in other countries [8,9]. However, African and Asian countries face most significant concern, as they experience the majority of human rabies cases [10].

The key to preventing human rabies lies in immunizing residents and travelers visiting these rabies-endemic territories [6,11]. Travelers

staying in Southeast Asia for extended periods have an increased risk of coming into contact with rabies-transmitting animals, ranging from 0.3% to 4% for each month of stay [12,13]. Even though Serbia is not considered as heavily affected by animal rabies, there is still a considerable health risk for its population, particularly for travelers returning from rabies-endemic regions [4].

To prevent clinical rabies, timely post-exposure prophylaxis (PEP) is crucial. In Serbia, this includes intensive wound washing and wound infiltration with rabies immunoglobulin (RIG) to remove and neutralize the virus after single or multiple transdermal bites or scratches and contamination of mucous membrane by licks/bites (WHO category III exposure) of any wild vertebrate. Accordingly, pre-exposure

<footnote><sup>a</sup> Department for Prevention of Rabies and Other Infectious Diseases, Pasteur Institute Novi Sad, Novi Sad 21000, Serbia</footnote>

<footnote><sup>b</sup> Department of Microbiology with Parasitology and Immunology, Faculty of Medicine, University of Novi Sad, Novi Sad 21000, Serbia</footnote>

<footnote><sup>c</sup> Department for Research & Monitoring of Rabies & Other Zoonoses, Pasteur Institute Novi Sad, 21000 Novi Sad, Serbia</footnote>

<footnote><sup>d</sup> Department of Microbiology, Pasteur Institute Novi Sad, 21000 Novi Sad, Serbia</footnote>

<footnote><sup>e</sup> Faculty of Public and One Health, School of Health Sciences, University of Thessaly, 24410 Karditsa, Greece</footnote>

<footnote><sup>f</sup> School of Environmental Sciences University of Guelph, Guelph, Ontario N1G 2W1, Canada</footnote>

<footnote><sup>g</sup> ANSES, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, Laboratoire de Santé Animale, Maisons-Alfort F-94700, France</footnote>

<footnote>* Corresponding author. Department for Prevention of Rabies and Other Infectious Diseases, Pasteur Institute Novi Sad, Novi Sad 21000, Serbia. E-mail address: pavle.banovic@mf.uns.ac.rs (P. Banović).</footnote>
<figure><img src="image_1.png" /></figure>

# Real-world evidence of rabies post-exposure prophylaxis in Serbia:
## Nation-wide observational study (2017–2019)

Pavle Banović<sup>a,b,*</sup>, Dragana Mijatović<sup>c</sup>, Verica Simin<sup>d</sup>, Nenad Vranješ<sup>c</sup>, Eleftherios Meletis<sup>e</sup>, Polychronis Kostoulas<sup>e</sup>, Dasiel Obregon<sup>f</sup>, Alejandro Cabezas-Cruz<sup>g</sup>

## ARTICLE INFO

### Keywords:

Rabies

Post-exposure prophylaxis

Essen regimen

Seroconversion

Rabies virus-neutralizing antibodies

## ABSTRACT

**Background:** Rabies remains a deadly zoonotic disease, primarily prevalent in Eastern European countries, with a significant global burden in Asia and Africa. Post-exposure prophylaxis (PEP) is critical to prevent clinical rabies. Serbia, a country with a relatively low animal rabies incidence, has been implementing a 4-dose Essen PEP regimen for 13 years. This real-world study aimed to assess the effectiveness of the 4-dose Essen regimen, considering demographic and clinical factors, after WHO Category III exposure.

**Method:** The study included 601 patients who received the 4-dose Essen PEP and 79 who received an additional 5th dose.

**Results:** Age emerged as a critical factor influencing seroconversion rates after the 4-dose regimen, with older individuals exhibiting lower RVNA titers. Logistic regression indicated a 3.18% decrease in seroconversion odds for each added year of age. The Cox proportional hazards mixed model highlighted age-related risks, with age groups 45–60 and 75–92 at the highest risk of non-seroconversion. Human Rabies Immune Globulin (HRIG) administration was associated with lower RVNA values after the 4-dose regimen, suggesting interference with vaccine immunogenicity among people who received larger doses of HRIG.

**Conclusions:** This study provides valuable real-world evidence for rabies PEP in a non-homogeneous population with potential comorbidities. The results underscore the importance of optimizing PEP strategies, particularly in older individuals, and reconsidering HRIG dosing to improve seroconversion rates.

## 1. Introduction

Rabies is a deadly disease that can be transmitted from animals to humans. It is caused by the rabies virus (RABV) and other related viruses from the *Lyssavirus* genus, Rhabdoviridae family [1]. In Europe, animal rabies is mainly found in Poland [2], Belarus [3], Ukraine [4,5], Russia, Moldova [6] and Romania [7], with sporadic cases reported in other countries [8,9]. However, African and Asian countries face most significant concern, as they experience the majority of human rabies cases [10].

The key to preventing human rabies lies in immunizing residents and travelers visiting these rabies-endemic territories [6,11]. Travelers

staying in Southeast Asia for extended periods have an increased risk of coming into contact with rabies-transmitting animals, ranging from 0.3% to 4% for each month of stay [12,13]. Even though Serbia is not considered as heavily affected by animal rabies, there is still a considerable health risk for its population, particularly for travelers returning from rabies-endemic regions [4].

To prevent clinical rabies, timely post-exposure prophylaxis (PEP) is crucial. In Serbia, this includes intensive wound washing and wound infiltration with rabies immunoglobulin (RIG) to remove and neutralize the virus after single or multiple transdermal bites or scratches and contamination of mucous membrane by licks/bites (WHO category III exposure) of any wild vertebrate. Accordingly, pre-exposure

<footnote><sup>a</sup> Department for Prevention of Rabies and Other Infectious Diseases, Pasteur Institute Novi Sad, Novi Sad 21000, Serbia</footnote>

<footnote><sup>b</sup> Department of Microbiology with Parasitology and Immunology, Faculty of Medicine, University of Novi Sad, Novi Sad 21000, Serbia</footnote>

<footnote><sup>c</sup> Department for Research & Monitoring of Rabies & Other Zoonoses, Pasteur Institute Novi Sad, 21000 Novi Sad, Serbia</footnote>

<footnote><sup>d</sup> Department of Microbiology, Pasteur Institute Novi Sad, 21000 Novi Sad, Serbia</footnote>

<footnote><sup>e</sup> Faculty of Public and One Health, School of Health Sciences, University of Thessaly, 24410 Karditsa, Greece</footnote>

<footnote><sup>f</sup> School of Environmental Sciences University of Guelph, Guelph, Ontario N1G 2W1, Canada</footnote>

<footnote><sup>g</sup> ANSES, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, Laboratoire de Santé Animale, Maisons-Alfort F-94700, France</footnote>

<footnote>* Corresponding author. Department for Prevention of Rabies and Other Infectious Diseases, Pasteur Institute Novi Sad, Novi Sad 21000, Serbia. E-mail address: pavle.banovic@mf.uns.ac.rs (P. Banović).</footnote>
immunization (PrEP) against rabies is of utmost importance for persons planning to visit and/or stay in rabies-endemic territories. PEP in Serbia is initiated after WHO category III exposure in urban environment if animal was showing aggressive behavior and is untraceable or dies within 10 days after a person was attacked. Vaccination is also essential, as it induces RABV-neutralizing antibodies (RVNA) that prevent the virus from entering peripheral nerves [14]. Successful PEP (i.e., sero-conversion) requires an RVNA titer of 0.5 IU/ml or higher [15]. The standard vaccination protocol, known as Essen regimen, involves one dose of vaccine each on days 0, 3, 7, 14, and 28 [16]. However, due to low compliance rates for the 5th dose (day 28) (e.g., see Ref. [17]), the World Health Organization (WHO) recommended a reduced Essen regimen that only includes the first four doses for healthy, fully immune competent individuals [18]. From 2018 WHO additionally simplified PEP, requiring only 3 visits to healthcare facility (i.e., on days 0,3,7 or 0, 7,21 for intradermal and intramuscular vaccine application, respectively) [19]. On the other hand U.S. Advisory Committee on Immunization Practices still recommend 4-dose PEP [20], pending more data to evaluate the 3 dose series endorsed by WHO.

A noninferiority clinical trial in 500 healthy adults showed that satisfactory RVNA titer can be achieved after 2-visit intradermal or intramuscular PrEP [21]. In 2018 position paper, WHO endorsed this regime, despite of the bias related to the age of study participants (all subjects were younger than 50 years) [15]. Recommendations for PrEP may be further simplified, since Jonker and Visser demonstrated that single vaccine dose can induce satisfactory RVNA titer in healthy adults [22].

While both WHO-recommended Essen regimens are considered as safe and effective, most of the data on immunization outcomes comes from studies involving healthy individuals under controlled clinical settings [23,24]. Little information exists about immunization response in other populations, such as those with comorbidities [25–31] or the elderly, whose immune response may be affected by immunosenescence [32,33]. Real-world evidence is needed to assess the effectiveness of vaccination regimes outside controlled settings, as clinical trials may have limitations and biases [34–36]. Hence, vaccination regimens require support from diverse situations that would be present in a real-world scenario, such as the ones previously reported where local patients and international travelers did not receive optimal rabies PEP [11,37–40] and as a consequence inappropriate antibody response is observed [24]. The number of international travelers older than 60 years who may require PEP is rising dramatically [41], therefore there is an objective need for assessment of outcome of standard immunization protocols outside controlled clinical settings, embracing the real-world evidence data.

In Serbia, patients requiring PEP have been administered the reduced Essen regimen for the past 13 years. Patients who did not achieve an RVNA titer ≥0.5 IU/ml two weeks after 4th dose (day 21) received the additional vaccine doses until RVNA titer ≥0.5 IU/ml is achieved [42].

This study aims to assess the effectiveness of reduced (4-dose) Essen regimen in patients of different genders and ages using real-world evidence. The study focuses on the seroconversion rate in patients vaccinated after WHO category III exposure in Serbia.

# 2. Material and methods

## 2.1. Study settings

In Serbia, rabies prophylaxis is carried out through 27 Anti-Rabies Stations (ARSs) and coordinated by the Pasteur Institute Novi Sad. The official national guideline during the study period [43], prescribed PEP using Purified Vero cell Rabies Vaccine (PVRV; Verorab®, Sanofi) administered intramuscularly in 4-dose Essen regime. The vaccination schedule includes shots on days 0, 3, 7 and 14 after exposure, as per WHO category III exposure criteria. If locally produced Human Rabies Immune Globulin (HRIG; Blood Transfusion Institute of Serbia,

Belgrade) was available in the ARS, it was recommended to be given along with the first vaccine dose, at a dosage of 20 IU per kg of patient body weight. HRIG was used to infiltrate possible RABV entrance points, while remaining volume (if any) was administered in gluteal region intramuscularly. All ARSs were instructed to avoid administration of HRIG and PVRV in the same extremity. If the HRIG was not locally available, only vaccine was administered according to previously described regimen (i.e. days 0, 3, 7 and 14).

Two weeks after the 4th vaccine shot, a blood sample was taken from the patient at the ARS and sent to Pasteur Institute Novi Sad for RVNA titer analysis using the Rapid Fluorescent Focus Inhibition test (RFFIT) [44]. If the RVNA titer was below 0.5 IU/ml, 4-dose PEP was considered as unsuccessful, and the ARS was instructed to administer a 5th vaccine shot. Two weeks later, another blood sample was collected from the patient at the ARS for a new RVNA titer analysis. This procedure was repeated until protective RVNA titer of ≥0.5 IU/ml was achieved.

The RFFIT used for quantification of RVNA is calibrated against WHO reference serum standard and undergoes routine validation through proficiency testing organized by the European reference laboratory for Rabies, The Nancy laboratory for rabies and wildlife, under the French Agency for Food, Environmental and Occupational Health Safety.

## 2.2. Study design

This retrospective observational study utilized medical documentation provided by ARS and/or patient themselves. It included basic demographic information (age, gender), immunization protocol data, HRIG administration data, vaccine lot number (vaccine potency), and RFFIT results (RVNA titer) obtained from the RFFIT database (Access, Microsoft Office 2013) maintained by the National Reference Laboratory for Rabies. The study focused on patients who underwent PEP between January 01, 2017 and December 31, 2019 (Fig. 1).

To be eligible for the analysis, patients needed to meet the following inclusion criteria: (i) completion of the PEP protocol (receipt of all prescribed vaccines) with RFFIT performed on their serum sample at least two weeks after the last PVRV vaccine, (ii) medical documentation provided by the local ARS upon completion of immunization, (iii) all PVRV vaccines used for PEP in one patient must be from the same LOT (having the same potency), and (iv) for cases involving HRIG administration, the applied dosage needed to be 20 IU/kg of body weight.

Data with parameters of interest were generated for all individuals who received 4 vaccines against rabies (4-dose Essen cohort). Additional records were generated for patients who did not reach an RVNA titer ≥0.5 IU/ml after the reduced Essen protocol, and these records formed the 5-dose Essen cohort. Both cohort's records were further analyzed and compared.

## 2.3. Statistical analysis

In this study, we conducted various statistical analyses using the R programming language [45] and several R-packages, namely "survival" [46], "coxe" [47], and "survminer" [48]. We began with standard descriptive summaries for all variables in the study, excluding multivariate and univariate outliers from the sample.

For determining the strongest predictors of seroconversion, we performed linear regression. Additionally, we investigated the correlation between continuous independent variables and RVNA titers after logarithmic transformation (Y = log(Y)) to understand their relationship. To identify the most influential factors for achievement of seroconversion, logistic regression was performed. Furthermore, we used individual continuous variables as dependent variables in a series of t-tests to assess any significant differences between patients who achieved seroconversion and those requiring extension of the PEP regimen with additional vaccine doses. For instance, we examined whether two groups with RVNA <0.5 IU/ml and RVNA ≥0.5 IU/ml after 4-dose PEP showed
immunization (PrEP) against rabies is of utmost importance for persons planning to visit and/or stay in rabies-endemic territories. PEP in Serbia is initiated after WHO category III exposure in urban environment if animal was showing aggressive behavior and is untraceable or dies within 10 days after a person was attacked. Vaccination is also essential, as it induces RABV-neutralizing antibodies (RVNA) that prevent the virus from entering peripheral nerves [14]. Successful PEP (i.e., sero-conversion) requires an RVNA titer of 0.5 IU/ml or higher [15]. The standard vaccination protocol, known as Essen regimen, involves one dose of vaccine each on days 0, 3, 7, 14, and 28 [16]. However, due to low compliance rates for the 5th dose (day 28) (e.g., see Ref. [17]), the World Health Organization (WHO) recommended a reduced Essen regimen that only includes the first four doses for healthy, fully immune competent individuals [18]. From 2018 WHO additionally simplified PEP, requiring only 3 visits to healthcare facility (i.e., on days 0,3,7 or 0, 7,21 for intradermal and intramuscular vaccine application, respectively) [19]. On the other hand U.S. Advisory Committee on Immunization Practices still recommend 4-dose PEP [20], pending more data to evaluate the 3 dose series endorsed by WHO.

A noninferiority clinical trial in 500 healthy adults showed that satisfactory RVNA titer can be achieved after 2-visit intradermal or intramuscular PrEP [21]. In 2018 position paper, WHO endorsed this regime, despite of the bias related to the age of study participants (all subjects were younger than 50 years) [15]. Recommendations for PrEP may be further simplified, since Jonker and Visser demonstrated that single vaccine dose can induce satisfactory RVNA titer in healthy adults [22].

While both WHO-recommended Essen regimens are considered as safe and effective, most of the data on immunization outcomes comes from studies involving healthy individuals under controlled clinical settings [23,24]. Little information exists about immunization response in other populations, such as those with comorbidities [25-31] or the elderly, whose immune response may be affected by immunosenescence [32,33]. Real-world evidence is needed to assess the effectiveness of vaccination regimes outside controlled settings, as clinical trials may have limitations and biases [34-36]. Hence, vaccination regimens require support from diverse situations that would be present in a real-world scenario, such as the ones previously reported where local patients and international travelers did not receive optimal rabies PEP [11,37-40] and as a consequence inappropriate antibody response is observed [24]. The number of international travelers older than 60 years who may require PEP is rising dramatically [41], therefore there is an objective need for assessment of outcome of standard immunization protocols outside controlled clinical settings, embracing the real-world evidence data.

In Serbia, patients requiring PEP have been administered the reduced Essen regimen for the past 13 years. Patients who did not achieve an RVNA titer ≥0.5 IU/ml two weeks after 4th dose (day 21) received the additional vaccine doses until RVNA titer ≥0.5 IU/ml is achieved [42].

This study aims to assess the effectiveness of reduced (4-dose) Essen regimen in patients of different genders and ages using real-world evidence. The study focuses on the seroconversion rate in patients vaccinated after WHO category III exposure in Serbia.

# 2. Material and methods

## 2.1. Study settings

In Serbia, rabies prophylaxis is carried out through 27 Anti-Rabies Stations (ARSs) and coordinated by the Pasteur Institute Novi Sad. The official national guideline during the study period [43], prescribed PEP using Purified Vero cell Rabies Vaccine (PVRV; Verorab®, Sanofi) administered intramuscularly in 4-dose Essen regime. The vaccination schedule includes shots on days 0, 3, 7 and 14 after exposure, as per WHO category III exposure criteria. If locally produced Human Rabies Immune Globulin (HRIG; Blood Transfusion Institute of Serbia,

Belgrade) was available in the ARS, it was recommended to be given along with the first vaccine dose, at a dosage of 20 IU per kg of patient body weight. HRIG was used to infiltrate possible RABV entrance points, while remaining volume (if any) was administered in gluteal region intramuscularly. All ARSs were instructed to avoid administration of HRIG and PVRV in the same extremity. If the HRIG was not locally available, only vaccine was administered according to previously described regimen (i.e. days 0, 3, 7 and 14).

Two weeks after the 4th vaccine shot, a blood sample was taken from the patient at the ARS and sent to Pasteur Institute Novi Sad for RVNA titer analysis using the Rapid Fluorescent Focus Inhibition test (RFFIT) [44]. If the RVNA titer was below 0.5 IU/ml, 4-dose PEP was considered as unsuccessful, and the ARS was instructed to administer a 5th vaccine shot. Two weeks later, another blood sample was collected from the patient at the ARS for a new RVNA titer analysis. This procedure was repeated until protective RVNA titer of ≥0.5 IU/ml was achieved.

The RFFIT used for quantification of RVNA is calibrated against WHO reference serum standard and undergoes routine validation through proficiency testing organized by the European reference laboratory for Rabies, The Nancy laboratory for rabies and wildlife, under the French Agency for Food, Environmental and Occupational Health Safety.

## 2.2. Study design

This retrospective observational study utilized medical documentation provided by ARS and/or patient themselves. It included basic demographic information (age, gender), immunization protocol data, HRIG administration data, vaccine lot number (vaccine potency), and RFFIT results (RVNA titer) obtained from the RFFIT database (Access, Microsoft Office 2013) maintained by the National Reference Laboratory for Rabies. The study focused on patients who underwent PEP between January 01, 2017 and December 31, 2019 (Fig. 1).

To be eligible for the analysis, patients needed to meet the following inclusion criteria: (i) completion of the PEP protocol (receipt of all prescribed vaccines) with RFFIT performed on their serum sample at least two weeks after the last PVRV vaccine, (ii) medical documentation provided by the local ARS upon completion of immunization, (iii) all PVRV vaccines used for PEP in one patient must be from the same LOT (having the same potency), and (iv) for cases involving HRIG administration, the applied dosage needed to be 20 IU/kg of body weight.

Data with parameters of interest were generated for all individuals who received 4 vaccines against rabies (4-dose Essen cohort). Additional records were generated for patients who did not reach an RVNA titer ≥0.5 IU/ml after the reduced Essen protocol, and these records formed the 5-dose Essen cohort. Both cohort's records were further analyzed and compared.

## 2.3. Statistical analysis

In this study, we conducted various statistical analyses using the R programming language [45] and several R-packages, namely "survival" [46], "coxe" [47], and "survminer" [48]. We began with standard descriptive summaries for all variables in the study, excluding multivariate and univariate outliers from the sample.

For determining the strongest predictors of seroconversion, we performed linear regression. Additionally, we investigated the correlation between continuous independent variables and RVNA titers after logarithmic transformation (Y = log(Y)) to understand their relationship. To identify the most influential factors for achievement of seroconversion, logistic regression was performed. Furthermore, we used individual continuous variables as dependent variables in a series of t-tests to assess any significant differences between patients who achieved seroconversion and those requiring extension of the PEP regimen with additional vaccine doses. For instance, we examined whether two groups with RVNA <0.5 IU/ml and RVNA ≥0.5 IU/ml after 4-dose PEP showed
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Study flowchart. PEP - Post-exposure prophylaxis; HRIG - Human rabies immunoglobulin; RVNA – Rabies virus-neutralizing antibodies; IU – international units. Flowchart generated using open source software draw. io (https://app.diagrams.net/) [62].</figcaption></figure>

statistically significant difference on ‘Age’ variable. To explore potential
differences between expected and observed frequencies of the categor-
ical variables in relation to RVNA titers (<0.5 IU/ml or ≥ 0.5 IU/ml after
4-dose PEP), we conducted a series of Chi-square tests.

For the primary objective of determining the strongest predictors of seroconversion, we employed a logistic regression model. A Cox mixed effects model, referred also as Cox proportional hazards mixed model was applied in our study to analyze the time until the event of interest (seroconversion) occurs. The examined independent variables included available demographic information (age, gender), HRIG administration, units of HRIG administered, and residency.

The model was applied to two scenarios (i) successful immunization (i.e., seroconversion): after a 4-dose Essen PEP (scenario I) (ii) and successful immunization (i.e., seroconversion), after an extended (5-dose) Essen PEP (scenario II). To visualize the duration until seroconversion occurred, Kaplan-Meier curves were constructed. Graphs were generated using GraphPad Prism 9 and RStudio softwares.

3. Results

3.1. Characteristics of study participants

In this study, from 847 patients, total 601 subjects were included,
generating a total of 680 records (i.e., 601 records 4-dose Essen PEP and
79 records of 5th vaccine administration) (Fig. 1). Patients who were
excluded from the study were (i) immunized with different vaccine lots,

(ii) did not finish immunization in Serbia, and/or (iii) did not report for serum sampling after 4-dose Essen PEP was finished. All patients included in this study underwent 4-dose Essen PEP (n = 601), while only those who failed to achieve seroconversion (i.e., RVNA titer <0.5 IU/ml) had an additional record of 5th dose administration (n = 79). Females constituted the majority in both 4-dose Essen cohort (325/601; 54.07%) and the group requiring a 5th vaccine (47/79; 59.49%). The mean age of patients who underwent reduced Essen PEP was 43.88 years (95% CI 42.1–45.6), while the mean age of patients requiring additional vaccine dose was 55.3 years (95% CI: 51.8–58.8).

3.2. Demographic factors associated with 4-dose and 5-dose essen PEP
outcome

Immunization success rates were assessed after administering 4-dose and 5-dose Essen PEP, resulting in 84.5% (508/601) and 97.46% (77/79) success rates, respectively. Only two patients required six anti-rabies vaccines for achieving a minimum RVNA titer of ≥0.5 IU/ml. These patients were males of 84 and 33 years old and had recently undergone chemotherapy before PEP initiation.

There were no significant differences in seroconversion frequency between male or female subjects who underwent either 4-dose or 5-dose Essen PEP ($\chi^2(1) = 0.027, p > 0.05$, and $\chi^2(1) = 0.11, p > 0.05$, respectively).

The patients who achieved seroconversion after 4-dose and 5-dose
Essen PEP had a mean age of 46.88 (95% CI: 42.13–45.63) and 54.75
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Study flowchart. PEP - Post-exposure prophylaxis; HRIG - Human rabies immunoglobulin; RVNA – Rabies virus-neutralizing antibodies; IU – international units. Flowchart generated using open source software draw. io (https://app.diagrams.net/) [62].</figcaption></figure>

statistically significant difference on ‘Age’ variable. To explore potential
differences between expected and observed frequencies of the categor-
ical variables in relation to RVNA titers (<0.5 IU/ml or ≥ 0.5 IU/ml after
4-dose PEP), we conducted a series of Chi-square tests.

For the primary objective of determining the strongest predictors of seroconversion, we employed a logistic regression model. A Cox mixed effects model, referred also as Cox proportional hazards mixed model was applied in our study to analyze the time until the event of interest (seroconversion) occurs. The examined independent variables included available demographic information (age, gender), HRIG administration, units of HRIG administered, and residency.

The model was applied to two scenarios (i) successful immunization (i.e., seroconversion): after a 4-dose Essen PEP (scenario I) (ii) and successful immunization (i.e., seroconversion), after an extended (5-dose) Essen PEP (scenario II). To visualize the duration until seroconversion occurred, Kaplan-Meier curves were constructed. Graphs were generated using GraphPad Prism 9 and RStudio softwares.

3. Results

3.1. Characteristics of study participants

In this study, from 847 patients, total 601 subjects were included,
generating a total of 680 records (i.e., 601 records 4-dose Essen PEP and
79 records of 5th vaccine administration) (Fig. 1). Patients who were
excluded from the study were (i) immunized with different vaccine lots,

(ii) did not finish immunization in Serbia, and/or (iii) did not report for serum sampling after 4-dose Essen PEP was finished. All patients included in this study underwent 4-dose Essen PEP (n = 601), while only those who failed to achieve seroconversion (i.e., RVNA titer <0.5 IU/ml) had an additional record of 5th dose administration (n = 79). Females constituted the majority in both 4-dose Essen cohort (325/601; 54.07%) and the group requiring a 5th vaccine (47/79; 59.49%). The mean age of patients who underwent reduced Essen PEP was 43.88 years (95% CI 42.1–45.6), while the mean age of patients requiring additional vaccine dose was 55.3 years (95% CI: 51.8–58.8).

3.2. Demographic factors associated with 4-dose and 5-dose essen PEP
outcome

Immunization success rates were assessed after administering 4-dose and 5-dose Essen PEP, resulting in 84.5% (508/601) and 97.46% (77/79) success rates, respectively. Only two patients required six anti-rabies vaccines for achieving a minimum RVNA titer of ≥0.5 IU/ml. These patients were males of 84 and 33 years old and had recently undergone chemotherapy before PEP initiation.

There were no significant differences in seroconversion frequency between male or female subjects who underwent either 4-dose or 5-dose Essen PEP ($\chi^2(1) = 0.027, p > 0.05$, and $\chi^2(1) = 0.11, p > 0.05$, respectively).

The patients who achieved seroconversion after 4-dose and 5-dose
Essen PEP had a mean age of 46.88 (95% CI: 42.13–45.63) and 54.75
years (95% CI: 50.71–58.8), respectively. Age was found to be a factor related to immunization success after 4-dose Essen PEP (Mann-Whitney, U = 31,762, *p* < 0.05) but not after receiving a 5th dose (Mann-Whitney, U = 158.5, *p* > 0.05). A weak, yet significant negative correlation between age and RVNA titer was observed in the 4-dose Essen group (*r* = −0.3524, *p* < 0.05) (Fig. 2A), while no significant correlation was found in the 5-dose Essen group (*r* = −0.146, *p* > 0.05) (Fig. 2B).

Logistic regression analysis revealed that age is a risk factor for

unsuccessful immunization with 4-dose Essen, with odds of seroconversion decreasing by 3.18% for each additional year of age (β = 0.031395; SE = 0.005973).

Furthermore, the Cox proportional hazards mixed model showed that age influences immunization success. Specifically, age was associated with absence of seroconversion after the 4-dose Essen (scenario I); −0.007 (95% CI -0.011; −0.003). Age groups were categorized in 15-years intervals and inputted into the model as categorical data. For

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Association of RVNA titer with age and administration of HRIG. RVNA titer values used for Spearman correlation analysis were log transformed in order to achieve normal distribution. (A) RVNA titer is significantly associated with age of patients immunized via reduced (4-dose) Essen PEP; (B) RVNA titer is not associated with age of patients immunized via 5-dose Essen PEP; (C) Seroconversion rate is significantly associated with HRIG administration during reduced Essen PEP. Patients who received HRIG with 1st vaccine dose more frequently failed to reach seroconversion (RVNA titer ≥0.5 IU/ml) compared to patients where HRIG was not administered; bars are representing range intervals; Table is presenting GMT and range of RVNA titers for cohorts with and without HRIG administration and corresponds to chart above(D) Seroconversion rate is not associated with HRIG administration during 5-dose Essen PEP; bars are representing range intervals; Table is presenting GMT and range of RVNA titers for cohorts with and without HRIG administration before and after 5th dose administration and corresponds to chart above. RVNA – rabies virus neutralizing antibodies, *p* < 0.05, ns – not significant.</figcaption></figure>
years (95% CI: 50.71–58.8), respectively. Age was found to be a factor related to immunization success after 4-dose Essen PEP (Mann-Whitney, U = 31,762, *p* < 0.05) but not after receiving a 5th dose (Mann-Whitney, U = 158.5, *p* > 0.05). A weak, yet significant negative correlation between age and RVNA titer was observed in the 4-dose Essen group (*r* = −0.3524, *p* < 0.05) (Fig. 2A), while no significant correlation was found in the 5-dose Essen group (*r* = −0.146, *p* > 0.05) (Fig. 2B).

Logistic regression analysis revealed that age is a risk factor for

unsuccessful immunization with 4-dose Essen, with odds of seroconversion decreasing by 3.18% for each additional year of age (β = 0.031395; SE = 0.005973).

Furthermore, the Cox proportional hazards mixed model showed that age influences immunization success. Specifically, age was associated with absence of seroconversion after the 4-dose Essen (scenario I); −0.007 (95% CI -0.011; −0.003). Age groups were categorized in 15-years intervals and inputted into the model as categorical data. For

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Association of RVNA titer with age and administration of HRIG. RVNA titer values used for Spearman correlation analysis were log transformed in order to achieve normal distribution. (A) RVNA titer is significantly associated with age of patients immunized via reduced (4-dose) Essen PEP; (B) RVNA titer is not associated with age of patients immunized via 5-dose Essen PEP; (C) Seroconversion rate is significantly associated with HRIG administration during reduced Essen PEP. Patients who received HRIG with 1st vaccine dose more frequently failed to reach seroconversion (RVNA titer ≥0.5 IU/ml) compared to patients where HRIG was not administered; bars are representing range intervals; Table is presenting GMT and range of RVNA titers for cohorts with and without HRIG administration and corresponds to chart above(D) Seroconversion rate is not associated with HRIG administration during 5-dose Essen PEP; bars are representing range intervals; Table is presenting GMT and range of RVNA titers for cohorts with and without HRIG administration before and after 5th dose administration and corresponds to chart above. RVNA – rabies virus neutralizing antibodies, *p* < 0.05, ns – not significant.</figcaption></figure>
the 4-dose regimen, all age categories were associated with absence of seroconversion, with two age groups (45–60 and 75–92) having the highest risk (Fig. 3A).

The mean age of patients who did not achieve seroconversion after 4-dose Essen PEP was 55.3 years (95% CI: 51.8–58.8). To predict whether

a patient will require 5th vaccine dose to achieve RVNA titer ≥0.5 IU/ml, we used the lower boundary of the 95% CI of age from the group of unsuccessfully immunized patients after 4-dose Essen PEP (i.e., 51 years of age). After implementing this threshold and stratifying the patients into <51 and ≥51 years of age groups, we found statistically significant

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 3. A) Kaplan–Meier curve of probability for unsuccessful immunization after reduced Essen PEP for specific patient age groups in the context of time. Cox proportional hazards mixed model identified age groups 45–60 and 75–92 to have the highest risk of unsuccessful immunization after 4-dose Essen PEP B) Kaplan–Meier curve of probability for unsuccessful immunization after reduced Essen PEP in the context of time according if HRIG was administrated. Cox proportional hazards mixed model identified significant difference in probability of patient achieving seroconversion (i.e. immunization being successful) depending if patient received HRIG as part of 4-dose Essen PEP.</figcaption></figure>
the 4-dose regimen, all age categories were associated with absence of seroconversion, with two age groups (45–60 and 75–92) having the highest risk (Fig. 3A).

The mean age of patients who did not achieve seroconversion after 4-dose Essen PEP was 55.3 years (95% CI: 51.8–58.8). To predict whether

a patient will require 5th vaccine dose to achieve RVNA titer ≥0.5 IU/ml, we used the lower boundary of the 95% CI of age from the group of unsuccessfully immunized patients after 4-dose Essen PEP (i.e., 51 years of age). After implementing this threshold and stratifying the patients into <51 and ≥51 years of age groups, we found statistically significant

<figure><img src="image_5.png" /><figcaption>Fig. 3. A) Kaplan–Meier curve of probability for unsuccessful immunization after reduced Essen PEP for specific patient age groups in the context of time. Cox proportional hazards mixed model identified age groups 45–60 and 75–92 to have the highest risk of unsuccessful immunization after 4-dose Essen PEP B) Kaplan–Meier curve of probability for unsuccessful immunization after reduced Essen PEP in the context of time according if HRIG was administrated. Cox proportional hazards mixed model identified significant difference in probability of patient achieving seroconversion (i.e. immunization being successful) depending if patient received HRIG as part of 4-dose Essen PEP.</figcaption></figure>
difference ($\chi^2(1) = 16.341, p < 0.0001$) in the frequency of patients who achieved seroconversion. In addition, there was significance in the residuals of the patients above and below 51 years who failed to achieve seroconversion (Bonferroni adjustment, $p < 0.05$), indicating that more people above 51 years failed to reach seroconversion after 4-dose Essen PEP.

### 3.3. Effect of HRIG and vaccine potency on 4-dose and 5-dose Essen PEP outcome

HRIG was administered to 456 cases (75.87%) as part of the 4-dose Essen protocol and 72 cases (72/79; 91.13%) as part of the 5-dose Essen protocol. Seroconversion was achieved in 83.55% (381/456) and 95.83% (69/72) of patients, respectively. Patients who achieved seroconversion (i.e., had RVNA titer $\ge$0.5 IU/ml) after the 4-dose Essen protocol received an average of 1345.62 IU (95% CI: 1275.6-1415.5) of HRIG, while those who achieved seroconversion after the 5-dose Essen protocol received an average of 1438 IU (95% CI: 1350.8-1535.5). Patients who failed to reach seroconversion after 4- and 5-dose Essen protocols (i.e., had RVNA titer $<0.5$ IU/ml) received a higher amount of HRIG: 1456 IU (95% CI: 1382.8-1529.9) and 1480 IU (95% CI: 153.5-2806.4), respectively. Patients who failed to reach seroconversion after the 4-dose Essen regimen received a significantly higher amount of HRIG compared to subjects who achieved seroconversion (Mann-Whitney; $U = 9812.5, p < 0.05$). In addition, patients who hadn't received HRIG in the 4-dose Essen cohort developed significantly higher RVNA titer values compared to those where HRIG was included (Mann-Whitney; $U = 74.5, p < 0.05$) (Fig. 2C). These differences were not observed in the 5-dose Essen cohort (Mann-Whitney; $U = 154.5, p > 0.05$ and $U = 142.5, p > 0.05$, respectively). Despite the observed differences in the 4-dose Essen cohort, no correlation emerged between specific RVNA titer values and the administered amount of HRIG in both the 4-dose and 5-dose Essen protocols ($r = -0.07052, p > 0.05$, and $r = -0.1461, p > 0.05$).

The Cox proportional hazards mixed model also revealed that the administration of HRIG was associated with unsuccessful immunization for the 4-dose Essen regimen ($-0.27 (-0.515; -0.024)$) (Fig. 3B).

Over a 3-year period, seven PVRV vaccine lots were used for PEP in Republic of Serbia (i.e., K1391-2: 3.6 IU/dose; L1262-2:10.5 IU/dose; M16661V: 6.5 IU/dose, N1G972V: 9.7 IU/dose, N1J313V: 9.2 IU/dose, P1C531V: 10.8 IU/dose, and P1D271V: 6.2 IU/dose). However, we found no relationship between vaccine lot and seroconversion rate in either the 4-dose ($\chi^2(5) = 10.02, p > 0.05$) or the 5-dose Essen protocol ($\chi^2(5) = 3.96, p > 0.05$). Additionally, there was no significant difference in vaccine potency between patients who achieved seroconversion and those who did not for both the 4-dose (Mann-Whitney; $U = 19,039, p > 0.05$) and 5-dose Essen protocols (Mann-Whitney; $U = 179, p > 0.05$). Furthermore, no correlation was found between vaccine potency and RVNA titer for both the 4-dose and 5-dose Essen protocols ($r = -0.007, p > 0.05$ and $r = 0.046, p > 0.05$, respectively).

## 4. Discussion

This study provides valuable insights into the seroconversion rate after PEP against rabies and examines factors that may influence the effectiveness of immunization in real-world setting within one European country. It is important to know that the data here come from non-homogenous cohort where various common comorbidities are expected (e.g., hypertension, diabetes mellitus, various hormonal dysbalances, etc.) making them different from majority of studies that focus on healthy individuals in controlled settings. Nevertheless, we believe that data from real-world settings are more representative and can be highly valuable for decision-making regarding PEP against rabies.

In our research on the Serbian population immunized against rabies, the seroconversion rates were found to be 84.5% and 97.46% after 4-dose and 5-dose Essen PEP, respectively. Patient gender and PVRV

vaccine potency were not found to be significant factors affecting seroconversion rate in 4- or 5-dose Essen PEP. Accordingly, current WHO recommendations of 4-dose intramuscular PEP [49] could be considered as inadequate for specific individuals within this representative cohort.

The patient's age was identified as one on the few factors that is related with humoral response during 4-dose Essen PEP. Older patients showed lower RVNA production, consistent with previous studies [26, 50-52]. Different age-specific conditions may be the reason for this phenomena, such as development of auto-antibodies to type I interferons, that may modulate the immune response to vaccines in older individuals [53-55].

Furthermore, our seroconversion rates differed from those reported in Indian patients [23], possible due to differences in sample size and age distribution between the two studies. To compare the two studies, let's first look at the participant demographics. The Indian study enrolled 70 subjects, divided into reduced and complete Essen PEP groups, with an average age of 37.86 years for the 4-dose Essen group and 34.91 years for the 5-dose Essen group [23]. In contrast, our study included 601 patients who received the 4-dose Essen PEP and 79 patients who received the 5th vaccine dose, with average ages of 48.88 and 54.75 years, respectively.

A recent publication acknowledged that older adults tend to have lower levels of antibodies compared to younger adults [56]. However, it's important to note that the antibody levels observed in the study were significantly higher than the 0.5 IU/mL threshold, and as a result, these findings were not considered clinical significant. In addition, there's a long-standing assumption that individuals who have received modern cell culture vaccines for rabies are not experiencing breakthrough infections. It's worth noting that the threshold of 0.5 IU/mL for RVNA titer is somewhat arbitrary, and it's possible that even lower levels of antibodies may still offer protection against rabies.

On the other hand, it's important to recognize that for cases classified as WHO Category III exposure to rabies, there isn't a requirement to confirm rabies exposure before initiating PEP. This makes it challenging to accurately determine the real-world incidence of breakthrough infections. Although the “Law on measures for early detection, diagnosis and prevention of Rabies in Serbia” (Official Gazette of Republic of Serbia No. 78/2009) provides detailed instructions for animal surveillance and analysis on rabies after WHO category III exposure, it is extremely challenging to form a cohort of patients bitten by proven rabid animals in order to quantify incidence of breakthrough infections.

Another important factor affecting seroconversion was the administration of HRIG, which was found to be related to lower RVNA values. This interference with vaccine immunogenicity was more pronounced in the 4-dose Essen PEP group, as previously reported [57,58]. Additionally, we observed that HRIG-related interference was greater among individuals with higher body weight, who consequently received larger doses of HRIG. Although the precise mehanism leading to this phenomenon is still not completely understood, interference has been linked with the half-life of administered anti-RABV antibodies. As it was described previously, titer of RVNA derived from passive immunization is peaking within first 4 days and declining progressively until day 21 [59,60], with interference period possibly lasting up to day 28 [58]. Therefore, 5th vaccine in the case 4-dose Essen PEP extension is facing no interference with antibodies administered via RIG, allowing patient to achieve expected RVNA titer values. Implementing WHO recommendations to adjust the amount of RIG administered may help mitigate this interference and reduce costs of PEP [49]. More precisely, it was recommended that amount of RIG administered should be adjusted to achieve the maximal infiltration of the wound and not to rely on a body weight as a parameter to determine the RIG volume necessary for each patient [61]. In this way, patient still receives effective passive immunization, while intramuscular administration of RIG is avoided and interference with rabies vaccine immunogenicity is reduced, as well as the costs and the amount of RIG required for PEP. The current PEP
difference ($\chi^2(1) = 16.341, p < 0.0001$) in the frequency of patients who achieved seroconversion. In addition, there was significance in the residuals of the patients above and below 51 years who failed to achieve seroconversion (Bonferroni adjustment, $p < 0.05$), indicating that more people above 51 years failed to reach seroconversion after 4-dose Essen PEP.

### 3.3. Effect of HRIG and vaccine potency on 4-dose and 5-dose Essen PEP outcome

HRIG was administered to 456 cases (75.87%) as part of the 4-dose Essen protocol and 72 cases (72/79; 91.13%) as part of the 5-dose Essen protocol. Seroconversion was achieved in 83.55% (381/456) and 95.83% (69/72) of patients, respectively. Patients who achieved seroconversion (i.e., had RVNA titer $\ge$0.5 IU/ml) after the 4-dose Essen protocol received an average of 1345.62 IU (95% CI: 1275.6-1415.5) of HRIG, while those who achieved seroconversion after the 5-dose Essen protocol received an average of 1438 IU (95% CI: 1350.8-1535.5). Patients who failed to reach seroconversion after 4- and 5-dose Essen protocols (i.e., had RVNA titer $<0.5$ IU/ml) received a higher amount of HRIG: 1456 IU (95% CI: 1382.8-1529.9) and 1480 IU (95% CI: 153.5-2806.4), respectively. Patients who failed to reach seroconversion after the 4-dose Essen regimen received a significantly higher amount of HRIG compared to subjects who achieved seroconversion (Mann-Whitney; $U = 9812.5, p < 0.05$). In addition, patients who hadn't received HRIG in the 4-dose Essen cohort developed significantly higher RVNA titer values compared to those where HRIG was included (Mann-Whitney; $U = 74.5, p < 0.05$) (Fig. 2C). These differences were not observed in the 5-dose Essen cohort (Mann-Whitney; $U = 154.5, p > 0.05$ and $U = 142.5, p > 0.05$, respectively). Despite the observed differences in the 4-dose Essen cohort, no correlation emerged between specific RVNA titer values and the administered amount of HRIG in both the 4-dose and 5-dose Essen protocols ($r = -0.07052, p > 0.05$, and $r = -0.1461, p > 0.05$).

The Cox proportional hazards mixed model also revealed that the administration of HRIG was associated with unsuccessful immunization for the 4-dose Essen regimen ($-0.27 (-0.515; -0.024)$) (Fig. 3B).

Over a 3-year period, seven PVRV vaccine lots were used for PEP in Republic of Serbia (i.e., K1391-2: 3.6 IU/dose; L1262-2:10.5 IU/dose; M16661V: 6.5 IU/dose, N1G972V: 9.7 IU/dose, N1J313V: 9.2 IU/dose, P1C531V: 10.8 IU/dose, and P1D271V: 6.2 IU/dose). However, we found no relationship between vaccine lot and seroconversion rate in either the 4-dose ($\chi^2(5) = 10.02, p > 0.05$) or the 5-dose Essen protocol ($\chi^2(5) = 3.96, p > 0.05$). Additionally, there was no significant difference in vaccine potency between patients who achieved seroconversion and those who did not for both the 4-dose (Mann-Whitney; $U = 19,039, p > 0.05$) and 5-dose Essen protocols (Mann-Whitney; $U = 179, p > 0.05$). Furthermore, no correlation was found between vaccine potency and RVNA titer for both the 4-dose and 5-dose Essen protocols ($r = -0.007, p > 0.05$ and $r = 0.046, p > 0.05$, respectively).

## 4. Discussion

This study provides valuable insights into the seroconversion rate after PEP against rabies and examines factors that may influence the effectiveness of immunization in real-world setting within one European country. It is important to know that the data here come from non-homogenous cohort where various common comorbidities are expected (e.g., hypertension, diabetes mellitus, various hormonal dysbalances, etc.) making them different from majority of studies that focus on healthy individuals in controlled settings. Nevertheless, we believe that data from real-world settings are more representative and can be highly valuable for decision-making regarding PEP against rabies.

In our research on the Serbian population immunized against rabies, the seroconversion rates were found to be 84.5% and 97.46% after 4-dose and 5-dose Essen PEP, respectively. Patient gender and PVRV

vaccine potency were not found to be significant factors affecting seroconversion rate in 4- or 5-dose Essen PEP. Accordingly, current WHO recommendations of 4-dose intramuscular PEP [49] could be considered as inadequate for specific individuals within this representative cohort.

The patient's age was identified as one on the few factors that is related with humoral response during 4-dose Essen PEP. Older patients showed lower RVNA production, consistent with previous studies [26, 50-52]. Different age-specific conditions may be the reason for this phenomena, such as development of auto-antibodies to type I interferons, that may modulate the immune response to vaccines in older individuals [53-55].

Furthermore, our seroconversion rates differed from those reported in Indian patients [23], possible due to differences in sample size and age distribution between the two studies. To compare the two studies, let's first look at the participant demographics. The Indian study enrolled 70 subjects, divided into reduced and complete Essen PEP groups, with an average age of 37.86 years for the 4-dose Essen group and 34.91 years for the 5-dose Essen group [23]. In contrast, our study included 601 patients who received the 4-dose Essen PEP and 79 patients who received the 5th vaccine dose, with average ages of 48.88 and 54.75 years, respectively.

A recent publication acknowledged that older adults tend to have lower levels of antibodies compared to younger adults [56]. However, it's important to note that the antibody levels observed in the study were significantly higher than the 0.5 IU/mL threshold, and as a result, these findings were not considered clinical significant. In addition, there's a long-standing assumption that individuals who have received modern cell culture vaccines for rabies are not experiencing breakthrough infections. It's worth noting that the threshold of 0.5 IU/mL for RVNA titer is somewhat arbitrary, and it's possible that even lower levels of antibodies may still offer protection against rabies.

On the other hand, it's important to recognize that for cases classified as WHO Category III exposure to rabies, there isn't a requirement to confirm rabies exposure before initiating PEP. This makes it challenging to accurately determine the real-world incidence of breakthrough infections. Although the “Law on measures for early detection, diagnosis and prevention of Rabies in Serbia” (Official Gazette of Republic of Serbia No. 78/2009) provides detailed instructions for animal surveillance and analysis on rabies after WHO category III exposure, it is extremely challenging to form a cohort of patients bitten by proven rabid animals in order to quantify incidence of breakthrough infections.

Another important factor affecting seroconversion was the administration of HRIG, which was found to be related to lower RVNA values. This interference with vaccine immunogenicity was more pronounced in the 4-dose Essen PEP group, as previously reported [57,58]. Additionally, we observed that HRIG-related interference was greater among individuals with higher body weight, who consequently received larger doses of HRIG. Although the precise mechanism leading to this phenomenon is still not completely understood, interference has been linked with the half-life of administered anti-RABV antibodies. As it was described previously, titer of RVNA derived from passive immunization is peaking within first 4 days and declining progressively until day 21 [59,60], with interference period possibly lasting up to day 28 [58]. Therefore, 5th vaccine in the case 4-dose Essen PEP extension is facing no interference with antibodies administered via RIG, allowing patient to achieve expected RVNA titer values. Implementing WHO recommendations to adjust the amount of RIG administered may help mitigate this interference and reduce costs of PEP [49]. More precisely, it was recommended that amount of RIG administered should be adjusted to achieve the maximal infiltration of the wound and not to rely on a body weight as a parameter to determine the RIG volume necessary for each patient [61]. In this way, patient still receives effective passive immunization, while intramuscular administration of RIG is avoided and interference with rabies vaccine immunogenicity is reduced, as well as the costs and the amount of RIG required for PEP. The current PEP
guideline in Serbia recommends dosing RIG according to body weight. Modifications may be made based on the WHO 2018 recommendation in cases where multiple PEPs are initiated in a single antirabies station during a shortage of RIG stock.

Our study's strengths lie in the large sample size and high degree of completeness, making the results representative of real-world data. However, some limitations should be acknowledged, such as the inability to assess the number and location of lesions requiring HRIG infiltration to identify local interference with vaccine particles, as well as the lack of evaluation of other PEP protocols. Although all patients reported no prior immunization against rabies, it is essential to note that the limitation of this observational study lies in the absence of RVNA titer verification for all subjects on day 0. This verification is crucial to substantiate that the ongoing PEP indeed serves as a primary immunization.

## 5. Conclusion

This study highlights age and HRIG administration as factors linked to seroconversion after 4-dose Essen PEP. We found that odds of immunization result in seroconversion is decreased by 3.18% for each additional year of patients age, as well as that age of 51 years is a threshold from which Serbian patient is expected to require 5th vaccine dose to achieve seroconversion. Understanding these factors can help optimize PEP strategies for rabies immunization in real-world settings. Future research should investigate the relationship between HRIG administration and vaccine immunogenicity in various PEP protocols (e. g, comparison of (i) 0,3,7,14; (ii) 0,3,7,28 and (iii) 0,3,7,14,28 schemes).

## Funding statement

Sanofi (RAB00058) provided data related to PVRV vaccines potency and funding to conduct the study. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Sanofi had the chance to review the manuscript prior of publication.

## CRediT authorship contribution statement

**Pavle Banović:** Writing – review & editing, Writing – original draft, Visualization, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Dragana Mijatović:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Verica Simin:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Nenad Vranješ:** Writing – review & editing, Resources, Data curation, Conceptualization. **Eleftherios Meletis:** Writing – review & editing, Visualization, Software, Methodology, Formal analysis, Data curation. **Polchronis Kostoulas:** Writing – review & editing, Validation, Methodology, Formal analysis, Data curation. **Dasiel Obregon:** Writing – review & editing, Visualization, Methodology, Formal analysis, Data curation, Conceptualization. **Alejandro Cabezas-Cruz:** Writing – review & editing, Validation, Super- vision, Software, Methodology, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## References

[1] Fooks AR, Shipley R, Markotter W, Tordo N, Freuling CM, Müller T, et al. Renewed public health threat from emerging lyssaviruses. Viruses 2021;13. https://doi.org/10.3390/v13091769.

[2] Resurgence of fox rabies in a formerly freed area in Poland | Rabies - Bulletin - Europe n.d. https://www.who-rabies-bulletin.org/news/resurgence-fox-rabies-for-merely-freed-area-poland (accessed December 4, 2022).

[3] Hanenko ON, Kolomiets ND, Tonko OV. Current trends in epidemiology of rabies in Belarus. Epidemiology and Vaccinal Prevention 2020;19:41–8. https://doi.org/10.31631/2073-3046-2020-19.5-41-8.

[4] Lojkić I, Simić I, Bedeković T, Kresić N. Current status of rabies and its eradication in eastern and southeastern europe. Pathogens 2021;10:742. https://doi.org/10.3390/pathogens10060742.

[5] Omelchenko HO, Avramenko NO, Kulynych SM, Polishchuk AA, Petrenko MO, Sysa PS, et al. Rabies virus spread in central and north-eastern regions of Ukraine. Med Weter 2022;78. https://doi.org/10.21521/mw.6605.6605-2022.

[6] Riccardi N, Giacomelli A, Antonello RM, Gobbi F, Angheben A. Rabies in Europe: an epidemiological and clinical update. Eur J Intern Med 2021;88:15–20. https://doi.org/10.1016/j.ejim.2021.04.010.

[7] Dascalu MA, Wasniewski M, Picard-Meyer E, Servat A, Daraban Bocaneti F, Tanase OI, et al. Detection of rabies antibodies in wild boars in north-east Romania by a rabies ELISA test. BMC Vet Res 2019;15:466. https://doi.org/10.1186/s12917-019-2209-x.

[8] Makovska IF, Krupinina TM, Nedosekov VV, Tsarenko TM, Novohatniy YA, Fahrion AS. Current issues and gaps in the implementation of rabies prevention in Ukraine in recent decades. Regulatory Mechanisms in Biosystems 2021;12:251–9. https://doi.org/10.15421/022134.

[9] Polupan I, Bezymennyi M, Gibaliuk Y, Drozhzhe Z, Rudoi O, Ukhovskiy V, et al. An analysis of rabies incidence and its geographic spread in the buffer area among orally vaccinated wildlife in Ukraine from 2012 to 2016. Front Vet Sci 2019;6.

[10] Gan H, Hou X, Wang Y, Xu G, Huang Z, Zhang T, et al. Global burden of rabies in 204 countries and territories, from 1990 to 2019: results from the Global Burden of Disease Study 2019. Int J Infect Dis 2023;126:136–44. https://doi.org/10.1016/j.ijid.2022.10.046.

[11] Meyerhoff P, Manekeller S, Saleh N, Boesecke C, Schlabe S, Wasmuth JC, et al. Rabies post-exposure prophylaxis in Germany - what are the challenges? Epidemiol Infect 2021;149:e119. https://doi.org/10.1017/S0950268821000601.

[12] Piyahanee W, Kittitrakul C, Lawpoolsri S, Gautret P, Kashino W, Tangkanakul W, et al. Risk of potentially rabid animal exposure among foreign travelers in Southeast Asia. PLoS Neglected Trop Dis 2012;6:e1852. https://doi.org/10.1371/journal.pntd.0001852.

[13] Piyahanee W, Shantavasinkul P, Phumratnanaprapin W, Udomchaisakul P, Wichianprasat P, Benjavongkulchai M, et al. Rabies exposure risk among foreign backpackers in Southeast Asia. Am J Trop Med Hyg 2010;82:1168–71. https://doi.org/10.4269/ajtmh.2010.09-0699.

[14] Kessels JA, Recuenco S, Navarro-Vela AM, Deray R, Vigilato M, Ertl H, et al. Pre-exposure rabies prophylaxis: a systematic review. Bull World Health Organ 2017; 95:210–219C. https://doi.org/10.2471/BLT.16.173039.

[15] Rabies vaccines: WHO position paper – April 2018 n.d. https://www.who.int/p/ publications-detail-redirect/who-wer9316 (accessed December 4, 2022).

[16] World Health Organization. WHO Expert Committee on Rabies [meeting held in Geneva from 24 to 30 September 1991] : eighth report. World Health Organization; 1992.

[17] Shankaraiah RH, Rajashekar RA, Veena V, Hanumanthaiah AND. Compliance to anti-rabies vaccination in post-exposure prophylaxis. Indian J Publ Health 2015; 59:58–60. https://doi.org/10.4103/0019-557X.152867.

[18] World Health Organization. WHO expert consultation on rabies: second report. World Health Organization; 2013.

[19] World Health Organisation. WHO expert consultation on rabies: third report. World Health Organisation; 2018.

[20] Rupprecht CE, Briggs D, Brown CM, Franka R, Katz SL, Kerr HD, et al. Use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent human rabies: recommendations of the advisory committee on immunization practices. MMWR Recomm Rep (Morb Mortal Wkly Rep) 2010;59:1–9.

[21] Soentjens P, Andries P, Aerssens A, Tsoumanis A, Ravinetto R, Heuninckx W, et al. Preexposure intradermal rabies vaccination: a noninferiority trial in healthy adults on shortening the vaccination schedule from 28 to 7 days. Clin Infect Dis 2019;68: 607–14. https://doi.org/10.1093/cid/ciy513.

[22] Jonker EFF, Visser LG. Single visit rabies pre-exposure priming induces a robust anamnestic antibody response after simulated post-exposure vaccination: results of a dose-finding study. J Trav Med 2017;24. https://doi.org/10.1093/jtm/tax033.

[23] Haradanahalli RS, Banerjee R, Kalappa MS, Narayana A, Annadani RR, Bilagumba G. Safety and immunogenicity of rabies vaccine as 4 - dose Essen Intramuscular regimen for post exposure prophylaxis: a non - randomized, comparative controlled study. Hum Vaccines Immunother 2021;17:2554–9. https://doi.org/10.1080/21645515.2021.1883388.

[24] Uwanyiligira M, Landry P, Genton B, de Valliere S. Rabies postexposure prophylaxis in routine practice in view of the new centers for disease control and prevention and world health organization recommendations. Clin Infect Dis 2012; 55:201–5. https://doi.org/10.1093/cid/cis384.

[25] Zhou L, Liu J, Li J, Liu X, Zhang J, Nie F, et al. A simulated, randomized study on the safety and immunogenicity of three consecutive commercial-scale batches of the CTN-1V strain human rabies vaccine to verify the inter-batch consistency. Hum Vaccines Immunother 2022:2138049. https://doi.org/10.1080/21645515.2022.2138049.

[26] Zhang L, Huang S, Cai L, Zhu Z, Chen J, Lu S, et al. Safety, immunogenicity of lyophilized purified vero cell cultured rabies vaccine administered in Zagreb and Essen regimen in post-exposure subjects: a post-marketing, parallel control clinical trial. Hum Vaccines Immunother 2021;17:2547–53. https://doi.org/10.1080/21645515.2021.1880200.
guideline in Serbia recommends dosing RIG according to body weight. Modifications may be made based on the WHO 2018 recommendation in cases where multiple PEPs are initiated in a single antirabies station during a shortage of RIG stock.

Our study's strengths lie in the large sample size and high degree of completeness, making the results representative of real-world data. However, some limitations should be acknowledged, such as the inability to assess the number and location of lesions requiring HRIG infiltration to identify local interference with vaccine particles, as well as the lack of evaluation of other PEP protocols. Although all patients reported no prior immunization against rabies, it is essential to note that the limitation of this observational study lies in the absence of RVNA titer verification for all subjects on day 0. This verification is crucial to substantiate that the ongoing PEP indeed serves as a primary immunization.

## 5. Conclusion

This study highlights age and HRIG administration as factors linked to seroconversion after 4-dose Essen PEP. We found that odds of immunization result in seroconversion is decreased by 3.18% for each additional year of patients age, as well as that age of 51 years is a threshold from which Serbian patient is expected to require 5th vaccine dose to achieve seroconversion. Understanding these factors can help optimize PEP strategies for rabies immunization in real-world settings. Future research should investigate the relationship between HRIG administration and vaccine immunogenicity in various PEP protocols (e. g, comparison of (i) 0,3,7,14; (ii) 0,3,7,28 and (iii) 0,3,7,14,28 schemes).

## Funding statement

Sanofi (RAB00058) provided data related to PVRV vaccines potency and funding to conduct the study. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Sanofi had the chance to review the manuscript prior of publication.

## CRediT authorship contribution statement

**Pavle Banović:** Writing – review & editing, Writing – original draft, Visualization, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Dragana Mijatović:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Verica Simin:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Nenad Vranješ:** Writing – review & editing, Resources, Data curation, Conceptualization. **Eleftherios Meletis:** Writing – review & editing, Visualization, Software, Methodology, Formal analysis, Data curation. **Polchronis Kostoulas:** Writing – review & editing, Validation, Methodology, Formal analysis, Data curation. **Dasiel Obregon:** Writing – review & editing, Visualization, Methodology, Formal analysis, Data curation, Conceptualization. **Alejandro Cabezas-Cruz:** Writing – review & editing, Validation, Super-vision, Software, Methodology, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## References

[1] Fooks AR, Shipley R, Markotter W, Tordo N, Freuling CM, Müller T, et al. Renewed public health threat from emerging lyssviruses. Viruses 2021;13. https://doi.org/10.3390/v13091769.

[2] Resurgence of fox rabies in a formerly freed area in Poland | Rabies - Bulletin - Europe n.d. https://www.who-rabies-bulletin.org/news/resurgence-fox-rabies-for-merely-freed-area-poland (accessed December 4, 2022).

[3] Hanenko ON, Kolomiets ND, Tonko OV. Current trends in epidemiology of rabies in Belarus. Epidemiology and Vaccinal Prevention 2020;19:41–8. https://doi.org/10.31631/2073-3046-2020-19.5-41-8.

[4] Lojkić I, Simić I, Bedeković T, Kresić N. Current status of rabies and its eradication in eastern and southeastern europe. Pathogens 2021;10:742. https://doi.org/10.3390/pathogens10060742.

[5] Omelchenko HO, Avramenko NO, Kulynych SM, Polishchuk AA, Petrenko MO, Sysa PS, et al. Rabies virus spread in central and north-eastern regions of Ukraine. Med Weter 2022;78. https://doi.org/10.21521/mw.6605. 6605-2022.

[6] Riccardi N, Giacomelli A, Antonello RM, Gobbi F, Angheben A. Rabies in Europe: an epidemiological and clinical update. Eur J Intern Med 2021;88:15–20. https://doi.org/10.1016/j.ejim.2021.04.010.

[7] Dascalu MA, Wasniewski M, Picard-Meyer E, Servat A, Daraban Bocaneti F, Tanase OI, et al. Detection of rabies antibodies in wild boars in north-east Romania by a rabies ELISA test. BMC Vet Res 2019;15:466. https://doi.org/10.1186/s12917-019-2209-x.

[8] Makovska IF, Krupinina TM, Nedosekov VV, Tsarenko TM, Novohatniy YA, Fahrion AS. Current issues and gaps in the implementation of rabies prevention in Ukraine in recent decades. Regulatory Mechanisms in Biosystems 2021;12:251–9. https://doi.org/10.15421/022134.

[9] Polupan I, Bezymennyi M, Gibaliuk Y, Drozhzhe Z, Rudoi O, Ukhovskiy V, et al. An analysis of rabies incidence and its geographic spread in the buffer area among orally vaccinated wildlife in Ukraine from 2012 to 2016. Front Vet Sci 2019;6.

[10] Gan H, Hou X, Wang Y, Xu G, Huang Z, Zhang T, et al. Global burden of rabies in 204 countries and territories, from 1990 to 2019: results from the Global Burden of Disease Study 2019. Int J Infect Dis 2023;126:136–44. https://doi.org/10.1016/j.ijid.2022.10.046.

[11] Meyerhoff P, Manekeller S, Saleh N, Boesecke C, Schlabe S, Wasmuth JC, et al. Rabies post-exposure prophylaxis in Germany - what are the challenges? Epidemiol Infect 2021;149:e119. https://doi.org/10.1017/S0950268821000601.

[12] Piyahanee W, Kittitrakul C, Lawpoolsri S, Gautret P, Kashino W, Tangkanakul W, et al. Risk of potentially rabid animal exposure among foreign travelers in Southeast Asia. PLoS Neglected Trop Dis 2012;6:e1852. https://doi.org/10.1371/journal.pntd.0001852.

[13] Piyahanee W, Shantavasinkul P, Phumratnanaprapin W, Udomchaisakul P, Wichianprasat P, Benjavongkulchai M, et al. Rabies exposure risk among foreign backpackers in Southeast Asia. Am J Trop Med Hyg 2010;82:1168–71. https://doi.org/10.4269/ajtmh.2010.09-0699.

[14] Kessels JA, Recuenco S, Navarro-Vela AM, Deray R, Vigilato M, Ertl H, et al. Pre-exposure rabies prophylaxis: a systematic review. Bull World Health Organ 2017; 95:210–219C. https://doi.org/10.2471/BLT.16.173039.

[15] Rabies vaccines: WHO position paper – April 2018 n.d. https://www.who.int/p/ publications-detail-redirect/who-wer9316 (accessed December 4, 2022).

[16] World Health Organization. WHO Expert Committee on Rabies [meeting held in Geneva from 24 to 30 September 1991] : eighth report. World Health Organization; 1992.

[17] Shankaraiah RH, Rajashekar RA, Veena V, Hanumanthaiah AND. Compliance to anti-rabies vaccination in post-exposure prophylaxis. Indian J Publ Health 2015; 59:58–60. https://doi.org/10.4103/0019-557X.152867.

[18] World Health Organization. WHO expert consultation on rabies: second report. World Health Organization; 2013.

[19] World Health Organisation. WHO expert consultation on rabies: third report. World Health Organisation; 2018.

[20] Rupprecht CE, Briggs D, Brown CM, Franka R, Katz SL, Kerr HD, et al. Use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent human rabies: recommendations of the advisory committee on immunization practices. MMWR Recomm Rep (Morb Mortal Wkly Rep) 2010;59:1–9.

[21] Soentjens P, Andries P, Aerssens A, Tsoumanis A, Ravinetto R, Heuninckx W, et al. Preexposure intradermal rabies vaccination: a noninferiority trial in healthy adults on shortening the vaccination schedule from 28 to 7 days. Clin Infect Dis 2019;68: 607–14. https://doi.org/10.1093/cid/ciy513.

[22] Jonker EFF, Visser LG. Single visit rabies pre-exposure priming induces a robust anamnestic antibody response after simulated post-exposure vaccination: results of a dose-finding study. J Trav Med 2017;24. https://doi.org/10.1093/jtm/tax033.

[23] Haradanahalli RS, Banerjee R, Kalappa MS, Narayana A, Annadani RR, Bilagumba G. Safety and immunogenicity of rabies vaccine as 4 - dose Essen Intramuscular regimen for post exposure prophylaxis: a non - randomized, comparative controlled study. Hum Vaccines Immunother 2021;17:2554–9. https://doi.org/10.1080/21645515.2021.1883388.

[24] Uwanyiligira M, Landry P, Genton B, de Valliere S. Rabies postexposure prophylaxis in routine practice in view of the new centers for disease control and prevention and world health organization recommendations. Clin Infect Dis 2012; 55:201–5. https://doi.org/10.1093/cid/cis384.

[25] Zhou L, Liu J, Li J, Liu X, Zhang J, Nie F, et al. A simulated, randomized study on the safety and immunogenicity of three consecutive commercial-scale batches of the CTN-1V strain human rabies vaccine to verify the inter-batch consistency. Hum Vaccines Immunother 2022:2138049. https://doi.org/10.1080/21645515.2022.2138049.

[26] Zhang L, Huang S, Cai L, Zhu Z, Chen J, Lu S, et al. Safety, immunogenicity of lyophilized purified vero cell cultured rabies vaccine administered in Zagreb and Essen regimen in post-exposure subjects: a post-marketing, parallel control clinical trial. Hum Vaccines Immunother 2021;17:2547–53. https://doi.org/10.1080/21645515.2021.1880200.
[27] Shen H, Wang Z, Yang B, Cai K, Jiang C, Xie R, et al. Immunogenicity and safety of purified vero cell-cultured rabies vaccine under Zagreb 2-1-1 or 5-dose Essen regimen in the healthy Chinese subjects: a randomized, double-blind, positive controlled phase 3 clinical trial. Hum Vaccines Immunother 2021;17:351-7. https://doi.org/10.1080/21645515.2020.1778408.

[28] Gholami A, Shirzadi M-R, Asouri M, Farahraj F, Mostafavi E, Gharibzadeh S, et al. Seroconversion after three doses of intramuscular rabies vaccine as a post-exposure treatment. Virus Res 2020;278:197883. https://doi.org/10.1016/j.viruses.2020.197883.

[29] Ambrozaitis A, Laiskonis A, Balciuniene L, Banzhoff A, Malerczyk C. Rabies post-exposure prophylaxis vaccination with purified chick embryo cell vaccine (PCECV) and purified Vero cell rabies vaccine (PVRV) in a four-site intradermal schedule (4-0-2-0-1-1): an immunogenic, cost-effective and practical regimen. Vaccine 2006; 24:4116-21. https://doi.org/10.1016/j.vaccine.2006.02.036.

[30] Li R, Huang L, Li J, Mo Z, He B, Wang Y, et al. A next-generation, serum-free, highly purified Vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab® when administered according to a post-exposure regimen in healthy children and adults in China. Vaccine 2013;31:5940-7. https://doi.org/10.1016/j.vaccine.2013.10.043.

[31] Li T, Wang X, Cheng H. Meta-analysis of immunogenicity and safety of human rabies vaccination under Zagreb and Essen regimens. J Comp Eff Res 2020;9: 459-68. https://doi.org/10.2217/cer-2019-0202.

[32] Chen WH, Kozlovsky BF, Effros RB, Grubeck-Loebenstein B, Edelman R, Sztain MB. Vaccination in the elderly: an immunological perspective. Trends Immunol 2009; 30:351-9. https://doi.org/10.1016/j.it.2009.05.002.

[33] Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to vaccines. Nat Immunol 2013;14:428-36. https://doi.org/10.1038/ni.2588.

[34] Bhatt A. Conducting real-world evidence studies in India. Perspect Clin Res 2019; 10:51-6. https://doi.org/10.4103/picr.picr_62_21.

[35] Kim H-S, Lee S, Kim JH. Real-world evidence versus randomized controlled trial: clinical research based on electronic medical records. J Kor Med Sci 2018;33:e213. https://doi.org/10.3346/jkms.2018.33.e213.

[36] Chodankar D. Introduction to real-world evidence studies. Perspect Clin Res 2021; 12:171-4. https://doi.org/10.4103/picr.picr_62_21.

[37] Saffar F, Heinemann M, Heitkamp C, Stelzl DR, Ramharter M, Schunk M, et al. Rabies post-exposure prophylaxis of international travellers - results from two major German travel clinics. Trav Med Infect Dis 2023;53:102573. https://doi.org/10.1016/j.tmaid.2023.102573.

[38] Tarantola A, Ly S, In S, Ong S, Peng Y, Heng N, et al. Rabies vaccine and rabies immunoglobulin in Cambodia: use and obstacles to use. J Trav Med 2015;22: 348-52. https://doi.org/10.1111/jtm.12228.

[39] Chomchay P, Khawplod P, Wilde H. Neutralizing antibodies to rabies following injection of rabies immune globulin into gluteal fat or deltoid muscle. J Trav Med 2000;7:187-8. https://doi.org/10.2310/7060.2000.00057.

[40] Gossner CM, Mailles A, Aznar I, Dimina E, Echevarría JE, Feruglio SL, et al. Prevention of human rabies: a challenge for the European union and the European economic area. Euro Surveill 2020;25:2000158. https://doi.org/10.2807/1560-7917.ES.2020.25.38.2000158.

[41] Ecarnot F, Maggi S, Michel J-P, Veronese N, Rossanese A. Vaccines and senior travellers. Front Aging 2021;2:677907. https://doi.org/10.3389/fragi.2021.677907.

[42] Muskinja N, Stankov S, Vujin D, Vranješ N, Lalosevic D. New immunization schedule with seroconversion testing. Med Data Rev 2010;2:279-82.

[43] Lončarević G. Stručno-metodološko uputstvo za sprovodenje obavezne i preporučene imunizacije stanovništva za 2017. godinu 2017:12-5.

[44] Stankov S, Vujin D, Simin V, Lazarević-Ivanc L, Stojadinović G, Vranjes N. Validation of the Rapid fluorescent focus inhibition test for rabies antibodies. Med Data Rev 2010;2:283-9.

[45] R: The R Project for Statistical Computing n.d. https://www.r-project.org/ (accessed July 19, 2023).

[46] Therneau TM. TL (original S->R port and R maintainer, Elizabeth A, Cynthia C. survival: survival Analysis 2023. until. 2009.

[47] Therneau TM. Coxme: mixed effects Cox models. 2022.

[48] Kassambara A, Kosinski M, Biecek P, Fabian S. survminer: drawing Survival Curves using "ggplot2". 2021.

[49] O'Brien KL, Nolan T. The WHO position on rabies immunization – 2018 updates. Vaccine 2019;37:A85-7. https://doi.org/10.1016/j.vaccine.2018.10.014.

[50] Li R, Li Y, Wen S, Wen H, Nong Y, Mo Z, et al. Immunogenicity and safety of purified chick-embryo cell rabies vaccine under Zagreb 2-1-1 or 5-dose Essen regimen in Chinese children 6 to 17 years old and adults over 50 years: a randomized open-label study. Hum Vaccines Immunother 2015;11:435-42. https://doi.org/10.4161/21645515.2014.994460.

[51] Mastroeni I, Vescia N, Pompa MG, Cattaruzza MS, Marini GP, Fara GM. Immune response of the elderly to rabies vaccines. Vaccine 1994;12:518-20. https://doi.org/10.1016/0264-410x(94)90310-7.

[52] Furuya-Kanamori L, Ramsey L, Manson M, Gilbert B, Lau CL. Intradermal rabies pre-exposure vaccination schedules in older travellers: comparison of immunogenicity post-primary course and post-booster. J Trav Med 2020;27: taaa006. https://doi.org/10.1093/jtm/taaa006.

[53] Hale BG. Autoantibodies targeting type I interferons: prevalence, mechanisms of induction, and association with viral disease susceptibility. Eur J Immunol 2023; 53:2250164. https://doi.org/10.1002/eji.202250164.

[54] Bastard P, Michailidis E, Hoffmann H-H, Chbihi M, Le Voyer T, Rosain J, et al. Auto-antibodies to type I IFNs can underlie adverse reactions to yellow fever live attenuated vaccine. J Exp Med 2021;218:e20202486. https://doi.org/10.1084/jem.20202486.

[55] Quiros-Roldan E, Sottini A, Signorini SG, Serana F, Tiecco G, Imberti L. Autoantibodies to interferons in infectious diseases. Viruses 2023;15:1215. https://doi.org/10.3390/v15051215.

[56] Xu C, Lau CL, Clark J, Rafferty AC, Mills DJ, Ramsey L, et al. Immunogenicity after pre- and post-exposure rabies vaccination: a systematic review and dose-response meta-analysis. Vaccine 2021;39:1044-50. https://doi.org/10.1016/j.vaccine.2021.01.023.

[57] Bernard M-C, Boudet F, Pineda-Pena A-C, Guinet-Morlot F. Inhibitory effect of concomitantly administered rabies immunoglobulins on the immunogenicity of commercial and candidate human rabies vaccines in hamsters. Sci Rep 2022;12: 6570. https://doi.org/10.1038/s41598-022-10281-1.

[58] Vodopija I, Sureau P, Smerdel S, Lafon M, Baklaić Z, Ijubićić M, et al. Interaction of rabies vaccine with human rabies immunoglobulin and reliability of a 2-1-1 schedule application for postexposure treatment. Vaccine 1988;6:283-6. https://doi.org/10.1016/0264-410x(88)90225-3.

[59] Peng J, Chen L, Zhu Z-G, Zhu Z-R, Hu Q, Fang Y. Effect of Corticosteroids on RVNA production of a patient with acute disseminated encephalomyelitis following rabies vaccination as well as administration of HRIG. Hum Vaccines Immunother 2014; 10:3622-6. https://doi.org/10.4161/21645515.2014.979621.

[60] Hanna K, Cruz MC, Mondou E, Corsi E, Vandeberg P. Safety and neutralizing rabies antibody in healthy subjects given a single dose of rabies immune globulin caprylate/chromatography purified. Clin Pharmacol 2018;10:79-88. https://doi.org/10.2147/CPAA.S166454.

[61] Schreuder I, De Pijper C, van Kessel R, Visser L, van den Kerkhof H. Dutch advisory committee on rabies. Abandon of intramuscular administration of rabies immunoglobulin for post-exposure prophylaxis in the revised guidelines in The Netherlands in 2018: cost and volume savings. Euro Surveill 2020;25:2000018. https://doi.org/10.2807/1560-7917.ES.2020.25.38.2000018.

[62] jgraph/drawio. Statements Ethical statement: this study was conducted in full accordance all applicable Pasteur Institute Novi Sad Research Policies and Serbian state laws and regulations, including the Patient Rights Law. 2023. The research was approved by the Ethical Committee of Medical Faculty Novi Sad (Approval number 01-39/72/1, dated 30th June 2021).
[27] Shen H, Wang Z, Yang B, Cai K, Jiang C, Xie R, et al. Immunogenicity and safety of purified vero cell-cultured rabies vaccine under Zagreb 2-1-1 or 5-dose Essen regimen in the healthy Chinese subjects: a randomized, double-blind, positive controlled phase 3 clinical trial. Hum Vaccines Immunother 2021;17:351-7. https://doi.org/10.1080/21645515.2020.1778408.

[28] Gholami A, Shirzadi M-R, Asouri M, Farahraj F, Mostafavi E, Gharibzadeh S, et al. Seroconversion after three doses of intramuscular rabies vaccine as a post-exposure treatment. Virus Res 2020;278:197883. https://doi.org/10.1016/j.viruses.2020.197883.

[29] Ambrozaitis A, Laiskonis A, Balciuniene L, Banzhoff A, Malerczyk C. Rabies post-exposure prophylaxis vaccination with purified chick embryo cell vaccine (PCECV) and purified Vero cell rabies vaccine (PVRV) in a four-site intradermal schedule (4-0-2-0-1-1): an immunogenic, cost-effective and practical regimen. Vaccine 2006; 24:4116-21. https://doi.org/10.1016/j.vaccine.2006.02.036.

[30] Li R, Huang L, Li J, Mo Z, He B, Wang Y, et al. A next-generation, serum-free, highly purified Vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab® when administered according to a post-exposure regimen in healthy children and adults in China. Vaccine 2013;31:5940-7. https://doi.org/10.1016/j.vaccine.2013.10.043.

[31] Li T, Wang X, Cheng H. Meta-analysis of immunogenicity and safety of human rabies vaccination under Zagreb and Essen regimens. J Comp Eff Res 2020;9: 459-68. https://doi.org/10.2217/cer-2019-0202.

[32] Chen WH, Kozlovsky BF, Effros RB, Grubeck-Loebenstein B, Edelman R, Sztain MB. Vaccination in the elderly: an immunological perspective. Trends Immunol 2009; 30:351-9. https://doi.org/10.1016/j.it.2009.05.002.

[33] Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to vaccines. Nat Immunol 2013;14:428-36. https://doi.org/10.1038/ni.2588.

[34] Bhatt A. Conducting real-world evidence studies in India. Perspect Clin Res 2019; 10:51-6. https://doi.org/10.4103/picr.picr_62_21.

[35] Kim H-S, Lee S, Kim JH. Real-world evidence versus randomized controlled trial: clinical research based on electronic medical records. J Kor Med Sci 2018;33:e213. https://doi.org/10.3346/jkms.2018.33.e213.

[36] Chodankar D. Introduction to real-world evidence studies. Perspect Clin Res 2021; 12:171-4. https://doi.org/10.4103/picr.picr_62_21.

[37] Saffar F, Heinemann M, Heitkamp C, Stelzl DR, Ramharter M, Schunk M, et al. Rabies post-exposure prophylaxis of international travellers - results from two major German travel clinics. Trav Med Infect Dis 2023;53:102573. https://doi.org/10.1016/j.tmaid.2023.102573.

[38] Tarantola A, Ly S, In S, Ong S, Peng Y, Heng N, et al. Rabies vaccine and rabies immunoglobulin in Cambodia: use and obstacles to use. J Trav Med 2015;22: 348-52. https://doi.org/10.1111/jtm.12228.

[39] Chomchay P, Khawplod P, Wilde H. Neutralizing antibodies to rabies following injection of rabies immune globulin into gluteal fat or deltoid muscle. J Trav Med 2000;7:187-8. https://doi.org/10.2310/7060.2000.00057.

[40] Gossner CM, Mailles A, Aznar I, Dimina E, Echevarría JE, Feruglio SL, et al. Prevention of human rabies: a challenge for the European union and the European economic area. Euro Surveill 2020;25:2000158. https://doi.org/10.2807/1560-7917.ES.2020.25.38.2000158.

[41] Ecarnot F, Maggi S, Michel J-P, Veronese N, Rossanese A. Vaccines and senior travellers. Front Aging 2021;2:677907. https://doi.org/10.3389/fragi.2021.677907.

[42] Muskinja N, Stankov S, Vujin D, Vranješ N, Lalosevic D. New immunization schedule with seroconversion testing. Med Data Rev 2010;2:279-82.

[43] Lončarević G. Stručno-metodološko uputstvo za sprovodenje obavezne i preporučene imunizacije stanovništva za 2017. godinu 2017:12-5.

[44] Stankov S, Vujin D, Simin V, Lazarević-Ivanc L, Stojadinović G, Vranjes N. Validation of the Rapid fluorescent focus inhibition test for rabies antibodies. Med Data Rev 2010;2:283-9.

[45] R: The R Project for Statistical Computing n.d. https://www.r-project.org/ (accessed July 19, 2023).

[46] Therneau TM. TL (original S->R port and R maintainer, Elizabeth A, Cynthia C. survival: survival Analysis 2023. until. 2009.

[47] Therneau TM. Coxme: mixed effects Cox models. 2022.

[48] Kassambara A, Kosinski M, Biecek P, Fabian S. survminer: drawing Survival Curves using "ggplot2". 2021.

[49] O'Brien KL, Nolan T. The WHO position on rabies immunization – 2018 updates. Vaccine 2019;37:A85-7. https://doi.org/10.1016/j.vaccine.2018.10.014.

[50] Li R, Li Y, Wen S, Wen H, Nong Y, Mo Z, et al. Immunogenicity and safety of purified chick-embryo cell rabies vaccine under Zagreb 2-1-1 or 5-dose Essen regimen in Chinese children 6 to 17 years old and adults over 50 years: a randomized open-label study. Hum Vaccines Immunother 2015;11:435-42. https://doi.org/10.4161/21645515.2014.994460.

[51] Mastroeni I, Vescia N, Pompa MG, Cattaruzza MS, Marini GP, Fara GM. Immune response of the elderly to rabies vaccines. Vaccine 1994;12:518-20. https://doi.org/10.1016/0264-410x(94)90310-7.

[52] Furuya-Kanamori L, Ramsey L, Manson M, Gilbert B, Lau CL. Intradermal rabies pre-exposure vaccination schedules in older travellers: comparison of immunogenicity post-primary course and post-booster. J Trav Med 2020;27: taaa006. https://doi.org/10.1093/jtm/taaa006.

[53] Hale BG. Autoantibodies targeting type I interferons: prevalence, mechanisms of induction, and association with viral disease susceptibility. Eur J Immunol 2023; 53:2250164. https://doi.org/10.1002/eji.202250164.

[54] Bastard P, Michailidis E, Hoffmann H-H, Chbihi M, Le Voyer T, Rosain J, et al. Auto-antibodies to type I IFNs can underlie adverse reactions to yellow fever live attenuated vaccine. J Exp Med 2021;218:e20202486. https://doi.org/10.1084/jem.20202486.

[55] Quiros-Roldan E, Sottini A, Signorini SG, Serana F, Tiecco G, Imberti L. Autoantibodies to interferons in infectious diseases. Viruses 2023;15:1215. https://doi.org/10.3390/v15051215.

[56] Xu C, Lau CL, Clark J, Rafferty AC, Mills DJ, Ramsey L, et al. Immunogenicity after pre- and post-exposure rabies vaccination: a systematic review and dose-response meta-analysis. Vaccine 2021;39:1044-50. https://doi.org/10.1016/j.vaccine.2021.01.023.

[57] Bernard M-C, Boudet F, Pineda-Pena A-C, Guinet-Morlot F. Inhibitory effect of concomitantly administered rabies immunoglobulins on the immunogenicity of commercial and candidate human rabies vaccines in hamsters. Sci Rep 2022;12: 6570. https://doi.org/10.1038/s41598-022-10281-1.

[58] Vodopija I, Sureau P, Smerdel S, Lafon M, Baklaić Z, Ijubićić M, et al. Interaction of rabies vaccine with human rabies immunoglobulin and reliability of a 2-1-1 schedule application for postexposure treatment. Vaccine 1988;6:283-6. https://doi.org/10.1016/0264-410x(88)90225-3.

[59] Peng J, Chen L, Zhu Z-G, Zhu Z-R, Hu Q, Fang Y. Effect of Corticosteroids on RVNA production of a patient with acute disseminated encephalomyelitis following rabies vaccination as well as administration of HRIG. Hum Vaccines Immunother 2014; 10:3622-6. https://doi.org/10.4161/21645515.2014.979621.

[60] Hanna K, Cruz MC, Mondou E, Corsi E, Vandeberg P. Safety and neutralizing rabies antibody in healthy subjects given a single dose of rabies immune globulin caprylate/chromatography purified. Clin Pharmacol 2018;10:79-88. https://doi.org/10.2147/CPAA.S166454.

[61] Schreuder I, De Pijper C, van Kessel R, Visser L, van den Kerkhof H. Dutch advisory committee on rabies. Abandon of intramuscular administration of rabies immunoglobulin for post-exposure prophylaxis in the revised guidelines in The Netherlands in 2018: cost and volume savings. Euro Surveill 2020;25:2000018. https://doi.org/10.2807/1560-7917.ES.2020.25.38.2000018.

[62] jgraph/drawio. Statements Ethical statement: this study was conducted in full accordance all applicable Pasteur Institute Novi Sad Research Policies and Serbian state laws and regulations, including the Patient Rights Law. 2023. The research was approved by the Ethical Committee of Medical Faculty Novi Sad (Approval number 01-39/72/1, dated 30th June 2021).