The Immunological Basis

for Immunization Series

Module 17:
Rabies

Immunization, Vaccines and Biologicals

The Immunological Basis
for Immunization Series

Module 17:
Rabies

Immunization, Vaccines and Biologicals

WHO Library Cataloguing-in-Publication Data

The immunological basis for immunization series: module 17: rabies.

(Immunological basis for immunization series ; module 17)

1.Rabies - immunology. 2.Rabies virus - immunology. 3.Rabies vaccines - therapeutic use. 4.Immunization. I.World Health Organization.
II.Series.

ISBN 978 92 4 150108 8 (NLM classification: WC 550)

© World Health Organization 2011

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia,
1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to repro-
duce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the above
address (fax: +41 22 791 4806; e-mail: permissions@who.int).
The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on
the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning
the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full
agreement.

The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary
products are distinguished by initial capital letters.

All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However,
the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation
and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use.
 The Department of Immunization, Vaccines and Biologicals
thanks the donors whose unspecified financial support
has made the production of this document possible.

This module was produced for Immunization, Vaccines and Biologicals, WHO, by:

Dr Deborah J Briggs, Executive Director

Global Alliance for Rabies Control
Manhattan Kansas, USA.

The Department of Immunization, Vaccines and Biologicals, WHO, also gratefully

acknowledges the kind assistance and contribution of the Department of Control of
Neglected Tropical Diseases in the preparation and review of this module.

Printed in February 2011

Copies of this publication as well as additional materials

on immunization, vaccines and biological may be requested from:

World Health Organization

Department of Immunization, Vaccines and Biologicals
CH-1211 Geneva 27, Switzerland
• Fax: + 41 22 791 4227 • Email: vaccines@who.int •

© World Health Organization 2010

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press,
World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 3264;
fax: +41 22 791 4857; email: bookorders@who.int). Requests for permission to reproduce or translate
WHO publications – whether for sale or for noncommercial distribution – should be addressed to
WHO Press, at the above address (fax: +41 22 791 4806; email: permissions@who.int).

The designations employed and the presentation of the material in this publication do not imply the
expression of any opinion whatsoever on the part of the World Health Organization concerning the
legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its
frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may
not yet be full agreement.

The mention of specific companies or of certain manufacturers’ products does not imply that they are
endorsed or recommended by the World Health Organization in preference to others of a similar nature
that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished
by initial capital letters.

All reasonable precautions have been taken by the World Health Organization to verify the information
contained in this publication. However, the published material is being distributed without warranty of
any kind, either expressed or implied. The responsibility for the interpretation and use of the material
lies with the reader. In no event shall the World Health Organization be liable for damages arising from
its use.

The named authors alone are responsible for the views expressed in this publication.

Printed by the WHO Document Production Services, Geneva, Switzerland

ii
 Contents

Abbreviations and acronyms..............................................................................................v

Preface............................................................................................................................... vii

1. Rabies virus and disease..........................................................................................1

1.1 Structure of the virus....................................................................................1
1.2 Classification.................................................................................................1
1.3 Pathology.......................................................................................................2
1.4 Epidemiology................................................................................................2

2. Immunity to rabies..................................................................................................3
2.1. Preventing clinical disease............................................................................3
2.2 Rabies vaccines..............................................................................................4
2.3 Response to immunization...........................................................................4
2.4 Role of passive immunity.............................................................................5
2.5 Routes of active immunization....................................................................6
2.6 Immune response in different populations..................................................6

3. Duration of immunity after immunization........................................................8

3.1 Development of immunity...........................................................................8
3.2 Duration of rabies virus-neutralizing antibody ........................................8
3.3 Anamnestic response...................................................................................10
3.4 Timeliness of routine booster vaccination.................................................11

4. Techniques to measure the immune response . ................................................12

4.1 Choosing the test to fit the purpose............................................................12
4.2 Virus neutralization assays.........................................................................12
4.3 Binding assays.............................................................................................13
4.4 Measuring cell-mediated immunity..........................................................13

5. Innocuity and efficacy of rabies biologicals.......................................................14

6. Future prospects....................................................................................................15

References.........................................................................................................................16

iii
iv
 Abbreviations and
acronyms

ABLV Australian bat lyssavirus

ARAV Aravan virus
CCV cell culture rabies vaccine
CNS central nervous system
dRIT direct rapid immunohistochemical test
DTP-IPV combined diphtheria, tetanus, whole cell pertussis and inactivated
poliomyelitis vaccine
DUVV Duvenhage virus
EBLV-1 European bat lyssavirus type 1
EBLV-2 European bat lyssavirus type 2
ELISA enzyme-linked immunosorbent assay
ERIG equine rabies immune globulin
FAT fluorescent antibody test
FAVN fluorescent antibody virus neutralization
GBS Guillain-Barré syndrome
HAART highly active antiretroviral therapy
HDCV human diploid cell vaccine
HIV human immunodeficiency virus
HRIG human rabies immune globulin
ICTC International Committee on Taxonomy of Viruses
ID intradermal
Ig immunoglobulin
IM intramuscular
IRKV Irkut virus
JEV Japanese encephalitis vaccine

v
KHUV Khujand virus
LBV Lagos bat virus
MHCII major histocompatibility complex class II
MNT mouse neutralization test
MOKV Mokola virus
NTV nerve tissue vaccines
PCECV purified chick embryo cell vaccine
PCR polymerase chain reaction
PDEV purified duck embryo cell vaccine
PEP post-exposure prophylaxis
PrEP pre-exposure vaccination
PVRV purified Vero cell rabies vaccine
RABV rabies virus
RFFIT rapid fluorescent focus inhibition test
RIDT rapid immunodiagnostic test
RIG rabies immune globulin
RNA ribonucleic acid
RTCIT rabies tissue culture infection test
RT-PCR reverse transcription-polymerase chain reaction
RVNA rabies virus-neutralizing antibody
SHIBV Shimoni bat virus
WCBV West Caucasian bat virus
WHO World Health Organization

vi
 Preface

This module is part of the series The Immunological Basis for Immunization,
which was initially developed in 1993 as a set of eight modules focusing on the vaccines
included in the Expanded Programme on Immunization (EPI)1. In addition to a general
immunology module, each of the seven other modules covered one of the vaccines
recommended as part of the EPI programme — diphtheria, measles, pertussis, polio,
tetanus, tuberculosis and yellow fever. The modules have become some of the most
widely used documents in the field of immunization.

With the development of the Global Immunization Vision and Strategy (GIVS)
(2005–2015) (http://www.who.int/vaccines-documents/DocsPDF05/GIVS_Final_
EN.pdf) and the expansion of immunization programmes in general, as well as the
large accumulation of new knowledge since 1993, the decision was taken to update
and extend this series.

The main purpose of the modules — which are published as separate disease/vaccine-
specific modules — is to give immunization managers and vaccination professionals
a brief and easily-understood overview of the scientific basis of vaccination, and also
of the immunological basis for the World Health Organization (WHO) recommendations
on vaccine use that, since 1998, have been published in the Vaccine Position Papers
(http://www.who.int/immunization/documents/positionpapers_intro/en/index.
html).

WHO would like to thank all the people who were involved in the development of
the initial Immunological Basis for Immunization series, as well as those involved in
its updating, and the development of new modules.

1
This programme was established in 1974 with the main aim of providing immunization for children
in developing countries.

vii
viii
 1. Rabies virus and disease

1.1 Structure of the virus

Rabies virus is an enveloped, rod-shaped virus containing a single-strand negative-sense

non-segmental ribonucleic acid (RNA) genome. It has a simple genome organization
that encodes five structural proteins: RNA polymerase (L); nucleocapsid protein
(N); phosphoprotein (P); matrix protein (M), and a surface glycoprotein (G). It is the
G that induces the production of rabies virus-neutralizing antibodies (RVNA) that
are the major immune effectors in protecting against infection with rabies virus (1,2).
The ribonucleoprotein complex consisting of the N, P, L, and negative-strand genomic
RNA has been reported to potentially play a role in the establishment of immunologic
memory and long-lasting immunity (3).

1.2 Classification

Rabies virus belongs to the genus Lyssavirus in the family Rhabdoviridae (1).
According to the International Committee on Taxonomy of Viruses (ICTV), as of
2009, 11 species were classified under the Lyssavirus genus (http://www.ictvonline.org).
The 11 species include: rabies virus (RABV), Lagos bat virus (LBV), Mokola virus
(MOKV), Duvenhage virus (DUVV), European bat lyssavirus type 1 (EBLV-1),
European bat lyssavirus type 2 (EBLV-2) and Australian bat lyssavirus (ABLV) (4,5),
Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV) and West Caucasian
bat virus (WCBV) (6–10). In addition, a new virus, Shimoni bat virus (SHIBV) was
reported in 2010, and needs further classification (11).

Besides rabies virus, viruses belonging to all other known lyssavirus genotypes have
been demonstrated (i.e.  ABLV,  DUVV,  EBLV,  MOKV),  or can all be expected 
(ARAV, KHUV, IRKV, WCBV and SHIBV) to cause a rabies-like lethal encephalitis
in humans.

Reports available on genetic classification of seven lyssavirus species/genotypes

(i.e. RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2 and ABLV) suggest that they
are divided into two phylogroups according to differences in genetic makeup,
serologic cross-reactivity and animal pathogenesis. Phylogroup I includes RABV,
DUVV, EBLV-1, EBLV-2 and ABLV, while phylogroup II includes LBV and MOKV
(6). The level of immunity against phylogroup II viruses induced by administering
the currently licensed rabies vaccines is dependent on how far the genetic makeup of
these viruses converges or diverges from that of RABV (7). The level of protection
provided against newly identified lyssaviruses by the currently available rabies vaccines
or immunoglobulins has, as yet, not been completely evaluated, and they may provide
reduced or no protection.

1
1.3 Pathology

Human rabies as a disease has the highest case-fatality rate ever reported (12).
Rabies virus infection can cause an acute progressive encephalopathy in a wide variety
of mammals, including humans, in which the disease almost invariably results in the
death of the host (12,13). After an exposure occurs, generally through infiltration of
virus-contaminated saliva from a rabid animal into a bite wound, or contact with mucous
membrane, the highly neurotropic rabies virus replicates in muscle tissue and enters
peripheral nerves, spreads by way of the peripheral nervous system to the spinal cord
and ascends to the brain. After dissemination within the central nervous system (CNS),
the virus spreads centrifugally from the CNS back along the nerves to various organs,
including the salivary glands, where it is emitted into the saliva and passed on to the
next victim, again usually through a bite wound or contamination of virus-carrying
saliva on to a mucous membrane (14).

1.4 Epidemiology

Rabies is an underreported disease that is present on every continent, except Antarctica,

with more than 55 000 human deaths occurring annually (15,16). Most human deaths
occur in Africa and Asia (15–18). Although all mammals are, to varying degrees,
susceptible to rabies, the primary reservoirs of the disease belong to the Orders
Carnivora and Chiroptera (i.e. dogs, foxes, jackals, coyotes, raccoon dogs, skunks,
raccoons, mongoose and bats) (13,19). Globally, over 98% of all human rabies deaths
occur following exposures to infected dogs. Millions of exposures to dogs occur
annually, with tens of thousands of human deaths resulting from cases of untreated
exposures (12,13,15). Human rabies, especially paralytic rabies (which may represent
as much as 30% of total clinical rabies presentations), is often misdiagnosed as other
encephalitic diseases, such as malaria or Guillain-Barré syndrome (GBS), thus masking
the true global burden of the disease (20,21,22).

2 WHO immunological basis for immunization series - Module 17: Rabies

 2. Immunity to rabies

2.1. Preventing clinical disease

Rabies is almost unique compared to other infections in that the development of

clinical disease following exposure to the virus is preventable, even in patients that
have not been previously vaccinated, through timely administration of post-exposure
prophylaxis (PEP). PEP, as recommended by WHO, comprises three components:
(a) wound treatment with cleansing, flushing and disinfection; (b) vaccine administration
over 28 to 90 days, depending on regimens and route of administration; (c) administration
of rabies immune globulin (RIG) with, or within the week which follows administration
of the first dose of vaccine in all category III (severe1) exposures (13,23). The outcome
of an exposure to rabies depends on many factors, including: the site and severity of
the exposure; the dose and variant (genotype or biotype) of virus inoculated into the
wound(s), and the timeliness of administering and adherence to WHO recommendations
for PEP (12,13,23). Both the innate (basic immune system inducing ‘non-specific’
resistance to disease) and adaptive (highly specialized, systemic cells and processes)
immune responses of a patient, are involved in securing protection against developing
rabies (24,25).

Besides wound treatment, whose action is mechanical and chemical, the primary
immunological objective of PEP is to neutralize and destroy rabies virus that was
inoculated into a victim’s body at the time of exposure. This needs to be achieved as
quickly as possible by increasing the amount of rabies virus neutralizing antibody
(RVNA) available to complete the task. Thus, it is critical for a protective immune
response to ensure that RVNA directed against the G of the virus is produced as soon
as possible (3,24). The level of RVNA is almost always high enough to be detected
between 7 to 14 days after primary vaccination (adaptive or active immunity) (41,42,43).
However, because human rabies is invariably fatal, the administration of rabies immune
globulin (RIG) (passive immunity) early in the vaccination regimen aims at providing
additional protection, especially for patients with severe and/or multiple wounds
(13,23,26,27,28).

1
Severe exposure is defined by WHO as single or multiple transdermal bites or scratches and licks
on broken skin, and contamination of mucous membrane with saliva.

3
2.2 Rabies vaccines

Since their development over three decades ago, cell culture- and embryonated
egg-based rabies vaccines (CCVs) have proved to be highly effective in preventing
human rabies, both when administered as pre-exposure vaccination (PrEP) and when
used in association with RIG for PEP (29). The production of CCVs represented a
significant advance, particularly over the first crude nerve tissue vaccines (NTV) for
rabies manufactured over a century ago using the brain material of infected animals (29).
It should be noted that all NTVs are reactogenic and the WHO has recommended
that they be replaced with CCVs (13). Several different cell substrates have been used
for the production of rabies vaccines, including Syrian baby hamster kidney cells,
human diploid cells, primary cell lines produced from embryonated chicken and duck
eggs, and continuous cell lines produced from Vero cells (29). Rabies vaccines produced
on Vero cells and primary cell lines originating from embryonated eggs have expanded
the safe use and availability of CCVs throughout the world. CCVs have also allowed a
broader use of vaccines for PrEP, so protecting those at constant risk of exposure (13,23).
Over the past two decades numerous data have been published proving the efficacy
and safety of CCVs (7,23,30–33). The cost of administering a 5-dose intramuscular (IM)
PEP regimen using CCVs is often beyond the financial capability of many persons
living in developing countries. Therefore, where budgetary limitations may deter the
use of CCVs for PEP, the WHO has recommended the administration of intradermal
(ID) PEP using CCVs that meet specific potency and immunological criteria (13).
Ongoing research specifically aimed at developing new, low cost and effective rabies
vaccines, could eventually reduce the global cost of preventing rabies (26).

2.3 Response to immunization

Early experiments designed to identify the components of the immune system responsible
for protection against rabies virus infection have proved that inactivated rabies vaccines
can induce the production of cytotoxic T-cells (34). Additional experimental evidence
investigating the role of cell-mediated immunity in mice confirmed the fact that
cytotoxic T- cells alone do not protect against rabies, as the depletion of CD8+ T-cells
had no effect on the resistance to disease nor on the survival rate of vaccinated animals
(3,24,35). Research indicates that inactivated rabies vaccines stimulate B-cells as well
as CD4+ cells using major histocompatibility complex class II (MHCII) mechanisms
and confer protection through the induction of an immune response including the
activation of lymphocytes, CD4+ antibody-secreting plasmocytes and neutralizing
antibodies that migrate into the nervous system parenchyma (3,24,35). The activation
of CD4+ T-lymphocytes ultimately results in the production of RVNAs that target and
destroy rabies virus, thus playing a major role in protecting victims exposed to rabies
against developing the disease (24,35,36).

The immune characteristics of both the humoral and cellular immune responses after
rabies vaccination was recently studied in 17 healthy patients and in five patients suffering
from a combined B- and T-cellular immunodeficiency (37). In all healthy patients the
enzyme-linked immunosorbent assay (ELISA) test results indicated that at one week
after primary vaccination there was a significant rise in the level of immunoglobulin
(Ig) M, and at two weeks after primary vaccination there was a significant rise in the
level of IgG (IgG1 and IgG3) and IgA. In the same study, after a booster vaccination
was administered, the level of IgG increased significantly faster (measured one week
after the booster dose) than it did after the primary series of doses was administered.

4 WHO immunological basis for immunization series - Module 17: Rabies

Overall, IgG1 is the major IgG subclass present after primary and booster rabies
vaccination. The five patients with a combined immunodeficiency, vaccinated using
the same protocol, showed a number of abnormalities in their humoral and cellular
immune responses.

Following inoculation of virulent rabies virus into animal models, the virus may either
replicate at the site of inoculation (usually in muscle tissue) or enter directly into the
peripheral nerves innervating the wound site without replication (38). Once rabies virus
enters the neurons neutralization could potentially be possible, although, according to
earlier research, it seems to be less likely (39). However, the pathogenesis of rabies is,
as yet, not completely defined and, because the administration of PEP has been effective
several days to months after an exposure has occurred, it is possible that RVNA can
occasionally clear rabies virus from the CNS.

Following administration of rabies vaccines, antibodies produced against other viral

proteins besides the G (specifically the N) have been detected in the sera of human
subjects. Published reports indicate that antibodies directed against the N do not
neutralize rabies virus, and therefore these specific ‘anti-N’ antibodies are unlikely to
play a major role in the development of protective humoral immunity. At present, the
role of non-neutralizing viral antibodies in providing immunity against disease is not
fully understood (25,40). There is no specific level of RVNA that is recognized as being
‘protective’ against rabies in humans, although WHO recommends an antibody level of
0.5 IU/mL as being proof of an adequate immune response after vaccination (13,23).

2.4 Role of passive immunity

Due to the critical role that RVNA plays in destroying by neutralizing rabies virus,
the level of protection against this disease can be enhanced through the immediate
administration of RIG into wounds inflicted by a rabid animal. RIG administration
delivers RVNA specifically targeted against rabies virus to the anatomical region
where it was injected during the trauma of the exposure. Convincing clinical evidence
proving that the administration of anti-rabies antiserum (in conjunction with vaccine)
into patients that were severely exposed to rabid animals reduced the risk of rabies,
was collected during a field study in Iran in 1954 (44). In this early study, different
doses of anti-rabies serum and/or vaccine were administered to 29 patients that had
received severe bite wounds from one rabid wolf. Of the 29 bite victims, 17 that had
incurred severe head wounds were treated as follows: five patients received two doses
of anti-rabies serum plus vaccine (all five patients survived); seven patients received
one dose of anti-rabies serum plus vaccine (one patient subsequently died of rabies),
and five patients received only vaccine (three patients subsequently died of rabies).
One six-year old patient that had received exceptionally deep head wounds including
a crushed skull received six doses of serum over a six-day period, plus vaccine,
and survived. The other patients involved in the exposure were bitten in the trunk and
legs and were administered either vaccine alone, or vaccine and serum. All of these
patients survived.

5
RIGs should be infiltrated into and around the wound sites of patients bitten by rabid
animals in order to neutralize rabies virus that may have been deposited in tissues
during an exposure (13). Human rabies immune globulin (HRIG) produced in human
subjects is administered at a dose of 20 IU/Kg of body weight, and equine rabies
immune globulin (ERIG) produced in horses is administered at a dose of 40 IU/Kg
of body weight. Unfortunately, due to the expense and lack of availability of RIGs,
not all patients that should receive passive immunity as part of PEP actually have access
to this life-saving product (13,15,45). Although the administration of vaccine alone will
save most patients, some patients will need to receive passive immunity immediately
in order to survive (46). Patients with bites into highly innervated regions such as the
head or hands, and those that have deep or multiple wounds, are the most vulnerable
and most in need of RIG (46,47). Specific recommendations for administration of RIG
as part of PEP is detailed elsewhere (http://www.who.int/rabies/human/en/index.
html) (13,23).

2.5 Routes of active immunization

The first CCVs, initially administered IM, were seen as the solution to replace early
reactogenic nerve tissue vaccines (NTVs) that normally induced a low or moderate
immune response (48). However, the high cost of CCVs relative to the cost of
NTVs, and the large number of patients that required PEP in canine rabies endemic
countries, initially curtailed the widespread use of CCVs. In an effort to alleviate the
situation by reducing the cost of CCVs without lowering the efficiency of the vaccine,
clinical trials were conducted to investigate the efficacy of ID regimens using a fraction
(60% to 80%) of the IM vaccine dose for PEP (49,50,51).

Over the past two decades, results published from several clinical trials have confirmed
the immunogenicity and efficacy of the ID route for rabies PEP which is currently
being used effectively in many Asian countries, including India, the Philippines,
Sri Lanka and Thailand (51,52,53). The ability of the ID route to induce an immunological
response results from the fact that the skin is an effective immune organ and vaccine
efficacy is enhanced when antigens are presented into the dermal layer (54,55,56).
Furthermore, the administration of antigens into the skin layer facilitates their exposure
to the numerous antigen-presenting cells, such as macrophages and dendritic cells, that
are present in higher numbers in skin than in muscle.

2.6 Immune response in different populations

CCVs are among the most immunogenic vaccines in the world, as is evidenced by
the very few reported human rabies deaths in patients that received prompt PEP
according to the WHO recommendations (25,28,57). Rabies vaccines are highly
immunogenic in almost every population, with perhaps the exception of patients
having very low CD4+ cells (58–61). In one study which investigated the immune
response of CCVs in selected populations of human immunodeficiency virus (HIV)
infected adults, only 57% of symptomatic HIV-infected patients with CD4+ counts
below 400 developed a measurable RVNA response above 0.5 IU/mL after receiving a
5-dose regimen of PEP intramuscularly on days 0, 3, 7, 14 and 30 (58). In another study,
10 HIV-infected adults with CD4+ counts between 25 and 472 were given a multi-site
PEP regimen whereby four doses of CCV were administered ID on days 0, 3 and 7, and
two doses of CCV were administered ID on days 28 and 90 (“4-4-4-0-2-2”) (60).

6 WHO immunological basis for immunization series - Module 17: Rabies

A lower than expected immune response was reported in all 10 patients; two of
the patients did not develop RVNA titres above 0.5 IU/ml by day 14, and one of
those patients did not develop RVNA above 0.5 IU/ml by day 30. In another study,
the immune response to a three-dose IM PrEP regimen was examined in 13 HIV-infected
children with CD4+ counts that were below normal, and compared to the immune
response in nine uninfected children (62). In this study, children that had fewer than
15% of the normal CD4+ cells had significantly lower RVNA titres when compared
with the control group and four of the 13 HIV-infected children failed to develop any
measurable RVNA. In a more recent study that examined the immune response after
vaccination with CCVs in HIV-infected patients receiving highly-active antiretroviral
therapy (HAART), slightly lower IgG and IgM titres were reported in older patients
infected with HIV (63). However, this study also reported that 63% of patients receiving
HAART still had measurable antibody titres five years after primary vaccination.
In another study evaluating more effective vaccination protocols in immunosuppressed
patients, two groups of HIV-positive subjects, one with CD4+ counts below 200 and
the other with CD4+ counts above 200, received a modified multiple eight-site series
of PEP consisting of eight intradermal injections on each of days 0, 3, 7, 14 and 30.
All subjects responded with titres above 0.5 IU/mL (64). PEP administered to children
that were exposed to rabies while receiving immunosuppressive therapy after solid
organ transplants, was also reported to be successful in all patients (65).

Infants and the elderly: The immune response to rabies vaccine in infants and the
elderly, without specific immunosuppressive conditions, is reported to be adequate.
In a report published that reviewed two studies examining the immune responses in
subjects of various ages, a reduction was observed in the level of RVNA after vaccination
in older individuals (66). In one of the reported studies, the immune response of
260 subjects between the age of 11 to 25 that received a six-dose PEP regimen was
compared to patients receiving the same regimen that were above the age of 50. In this
study, 52% of the adults above 50 years of age had significantly lower RVNA titres after
PEP compared to the younger cohort (67). In another study involving 875 patients aged
between 2–74 years of age that received either PEP or PrEP, no significant difference in
the production of RVNA compared to either age or sex was reported (68). The immune
response to rabies PEP was also reported to be highly immunogenic in children with
confirmed malnutrition, between Grade I and Grade IV (41).

Patients taking chloroquine for anti-malarial treatment: The administration of

rabies vaccine by the ID route has been reported to produce reduced titres in patients
taking chloroquine for anti-malarial treatment and, for this reason, vaccines should be
administered to this group of patients using the IM route (13,69,70).

Pregnant women: Rabies PEP is not counterindicated for pregnant women and is
immunogenic, safe and highly efficacious in this population (13). Rabies PEP should
never be withheld from pregnant women as it is a life-saving vaccine. No reported risk
of abortion, and no other harm to the fetus, has been reported due to administration
of PEP with CCV in pregnant women (71,72).

7
 3. Duration of immunity after
immunization

3.1 Development of immunity

The development of immunological memory after immunization with CCVs is a

critical component in the establishment of long-lasting immunity against rabies in
humans (3). Of the millions of persons that have received CCVs, less than a handful of
vaccination failures have been reported, all of which occurred in developing countries,
and most of which involved deviations from the WHO recommended PEP protocol
(25,28). Although one human death has been reported in a person that was previously
vaccinated with a CCV and subsequently exposed to a rabid puppy (70), this patient
did not seek, nor was she given the WHO recommended PEP booster series after
the exposure occurred. The results of several clinical trials and retrospective studies
have been published providing evidence that CCVs provide long-lasting immunity to
rabies.

3.2 Duration of rabies virus-neutralizing antibody

The measurement of RVNAs is the most convenient method of confirming an

immunological response after rabies PrEP or PEP. The relationship between the
number of doses a patient receives during the initial vaccination (PrEP or PEP) and the
longevity of circulating RVNA has been examined in several studies. In one retrospective
study, a Kaplan-Meier survival analysis was used to evaluate the longevity of antibody
in 875 patients that received either a primary three-dose (IM or ID) PrEP series or
a five-dose IM PEP series of human diploid cell vaccine (HDCV) (68). The study
reported no significant difference between the number of doses of vaccine a patient
received and the length of time after initial vaccination that RVNA could be detected.
In this study, no booster dose of vaccine was administered after the primary series,
and blood samples from patients were tested at various time intervals up to nine years
after primary vaccination. Circulating RVNA was detectable for a longer period of time
in patients that were vaccinated IM as opposed to patients that had been vaccinated by
the ID route, with approximately 80% of patients that had received vaccination by the
IM route still having detectable RVNA titres nine years after primary vaccination.

8 WHO immunological basis for immunization series - Module 17: Rabies

The longevity of the humoral immune response was also evaluated in 18 patients that
had received their primary series of PrEP or PEP using HDCV or purified chick embryo
cell vaccine (PCECV) from two to 14 years previously (73). The patients in this study
did not receive a booster vaccination between their initial series and the subsequent
drawing of blood that was analyzed for the presence of RVNA. All patients in the
study had detectable RVNA titres up to 14 years after having received their initial
vaccination. In another study, levels of RVNA were evaluated in 58 patients that received,
more than five years previously, PEP using HDCV, purified Vero cell rabies vaccine
(PVRV), purified duck embryo cell vaccine (PDEV) or PCECV by either the
Essen IM or Thai Red Cross ID regimen (74). All patients had detectable RVNA at the
time their blood was drawn. In a more recent study examining the longevity of antibody
and the effect of booster vaccination in 118 patients aged 16 to 78 years and vaccinated
five to 21 years previously with either HDCV or PVRV, all patients had detectable
antibody titres when they were tested prior to being given a booster dose of vaccine (75).
Finally, a study conducted in 29 travellers who had received their initial PreP using
HDCV by the ID route reported long-lasting immunity in patients that had received
their primary vaccination between two and >10 years previously (76).

In addition to published data delineating the extended duration of circulating RVNA

in patients that received only a primary PrEP or PEP vaccination series without an
additional booster vaccination at one year post-primary vaccination, studies have
reported long-lasting RVNA in patients that have received a primary series of PrEP
followed by one booster one year later. In one study, 312 subjects were followed for
10 years after having received either a 2-dose or 3-dose PrEP regimen, with either
HDCV or PVRV, and one booster dose of vaccine one year later (77). The results
indicate that approximately 96% of all subjects that received the 3-dose PrEP regimen
followed by one dose of vaccine one year later still had measurable RVNA 10 years
after having received their initial series. Similar results were reported in another
study where 10 subjects that had received their initial PrEP series with PCECV
14 years earlier were administered a booster dose one year later (78). In another study
conducted in 72 Vietnamese children, half of the children received a 3-dose series of a
combined diptheria, tetanus, whole cell pertussis and inactivated poliomyelitis vaccine
(DTP-IPV) along with three doses of PVRV given at two and four months and one year,
and the other half of the children received only DTP-IPV (33). Results from this study
indicate that rabies vaccines had no effect on the long-term antibody levels of diptheria
and poliomyelitis, and the majority of children continued to have measurable RVNA
titres throughout the five-year follow-up study. Similarly, a study was conducted in
200 Thai children that were vaccinated with PCECV in either a 2- or 3-dose IM or ID
PreP regimen concommitent with Japanese encephalitis vaccine (JEV), followed by
a booster dose of PCECV either IM or ID (as per the original route of vaccination)
one year later, and a booster dose of JEV (79). Three years after primary vaccination,
all children that received their initial PrEP series by the IM route or that received a
3-dose PrEP ID regimen still had detectable RVNA.

9
3.3 Anamnestic response

Two of the arguments in favour of administering PrEP to persons at risk of contracting

rabies are that if these persons are subsequently exposed to rabies:

1) a short series of booster vaccinations will elicit a rapid anamnestic response thus
reducing the number of doses of vaccine and visits required for a full PEP;
2) RIG is not required (13).

Several published clinical trials provide data confirming that a previously vaccinated
person will respond to one or more booster doses of rabies vaccine even if the initial
series of PrEP or PEP was administered several years previously; regardless of whether
the initial vaccination regimen was administered IM or ID; regardless of whether they
are boosted using the ID or IM route, and independent of whether the previously
vaccinated person has detectable RVNA or not (74,75,80,81,82). A three-year study
conducted in 194 subjects who initially received one, two, or three doses of HDCV
administered either by the IM or ID route, and were boosted six to 24 months later
with one dose of HDCV administered by the ID or IM route, reported the highest
titres and longest lasting antibodies in the subjects that had received an initial 3-dose
vaccination series (ID or IM) (80). All subjects in this study, regardless of whether they
had received one additional dose of vaccine ID or IM, had an anamnestic response when
boosted 6, 12, or 24 months later.

Another study reported that an anamnestic response occurred in 76 individuals

initially vaccinated with HDCV by the ID route and then boosted two years later
with one ID dose of HDCV (81). The anamnestic response occurred in all individuals,
regardless of whether the subjects had a detectable antibody titre just prior to the
administration of the booster, or not. Similar results were reported in a study where
29 travellers were initially vaccinated with a 3-dose ID HDCV regimen and boosted with
one IM dose of HDCV 12 to 14 months later (82). In this study, all persons developed
an anamnestic response even though some did not have detectable titres at the time that
they were boosted. In another study, the immune response of 57 patients vaccinated
for PEP, either by the 5-dose Essen IM regimen or the ID Thai Red Cross regimen,
were evaluated for a subsequent anamnestic response after receiving a booster vaccination
(74). In this study, patients were vaccinated 5–10 years previously with HDCV,
PCECV, PVRV or PDEV, and titres were evaluated after patients were boosted with
two ID doses of PDEV. All patients developed an anamnestic response after boosters
were administered, and there was no significant difference in the antibody level in
patients that had received vaccination 5–10 years earlier and those that had been
vaccinated more than 10 years previously. In another study, the immunological response
was examined in 118 patients that had received primary PEP or PrEP with HDCV
or PVRV five to 21 years earlier and were boosted with two ID doses of PVRV to
determine if they would mount an anamnestic response (75). In this study, all patients
vaccinated up to 21 years previously developed an immunological response with no
significant difference in the level of titres in patients that received PrEP or PEP, nor in
the length of time since their initial vaccination was administered.

10 WHO immunological basis for immunization series - Module 17: Rabies

3.4 Timeliness of routine booster vaccination

Due to the fact that rabies is virtually 100% fatal once clinical symptoms are
evident, and because until recently no long-term immunity studies were available,
the recommendations for timely routine booster doses of rabies vaccine after a
primary series has varied from one to five years. However, several recently-published
clinical trials have shown that individuals that have received an initial 3- to 5-dose
series of rabies CCVs will have long-term immunity lasting for decades (74,75,76,82).
These published data indicate that individuals that received their primary series up to
21 years previously will elicit a good anamnestic response after booster vaccination.

As mentioned earlier, individuals vaccinated with a CCV will respond to a booster

vaccination regardless of whether the vaccinated individual had measurable antibody
present or not at the time that the booster was administered (81,82,83). A recent survival
case in which a human patient that had been unknowingly given a transplanted liver
from a donor that was later diagnosed as having rabies provides an indication as to the
efficacy of rabies vaccines (84). The patient that received the infected liver survived,
whereas the recipients of the two kidneys and pancreas from the infected donor died
of rabies encephalitis within three weeks following transplant. Further investigation
revealed that the liver recipient had received rabies vaccination as a child.

11
 4. Techniques to measure the
immune response

4.1 Choosing the test to fit the purpose

Numerous assays are available to test for the presence of rabies virus in the tissues of
infected mammals, and to confirm evidence of a humoral or cellular immune response
after exposure to rabies viral antigens (85–89,96). Ultimately, the intended purpose
of an assay and the precision of the data required should be the determining factors
when choosing a testing procedure. For example, confirming herd immunity after
oral vaccination in animals generally does not require the same level of accuracy as
does evaluating the immunogenicity of a new rabies vaccine for humans, or when
serology testing is employed as part of the diagnostic workup for human rabies
patients (90,91,92). The importance of the level of quality assurance associated with
conducting any assay for evaluating an immune response for diagnoses of a patient,
or after immunization, or for identifying rabies antigen in tissue samples, cannot be
overstated, due to the consequences associated with a misdiagnosis (93). To summarize,
the sensitivity and specificity of an assay, the accuracy and precision required by the
investigator or clinician, the laboratory facilities that are available, and the purpose of
the data to be collected all need to be critically evaluated before testing of a sample’s
status is initiated (92).

4.2 Virus neutralization assays

Virus-neutralization assays are among the most widely-used methods to detect

the presence of antibody to the rabies virus. RVNAs are not only responsible
for protection against rabies, the presence of RVNA in serum is seen as a reliable
indicator of active immunization after vaccination (13,89). The rapid fluorescent focus
inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test
are both in vitro virus-neutralization assays. Both the RFFIT and the FAVN test are
equivalent when conducted under good laboratory practices and both are considered
to be the most efficient methods for accurate measurement of RVNA (13,90,94,95).
A third virus-neutralization assay, the mouse neutralization test (MNT), is an in vivo
method to measure RVNA that is still utilized in some laboratories that lack the capacity
to conduct in vitro tests (96). Methodologies for all three virus-neutralization assays
are published elsewhere (85,86,96,97,98). Virus-neutralization assays are valuable tools
that can confirm the presence of protective antibodies specifically targeted against
rabies virus, but they are also highly complex tests to perform and must be conducted
by experienced personnel in a high containment facility (92,93). It is advisable for
diagnostic laboratories performing either one or both of these types of RVNA assays
to participate in an established quality assurance programme (92).

12 WHO immunological basis for immunization series - Module 17: Rabies

4.3 Binding assays

The ELISA is the most frequently used binding assay available, with numerous
published protocols and professionally marketed ELISA kits available to detect
rabies antibodies (99–103). The specificity of the ELISA is dependent upon the choice
of the target antigen used in the test — whole virus or purified viral proteins.
Antibodies detected in an ELISA do not necessarily have a neutralizing function (92).
Published reports indicate that cross-reactivity, potentially leading to false positives,
may increase in ELISA assays that employ whole virus rather than purified G as the
target antigen (99,103). Several studies have been published comparing results from
serum samples tested by various ELISA techniques, and by the RFFIT or FAVN test,
with mixed results (89,102,104,105). Newer ELISA protocols and ELISA kits with
increased specificity, such as the PLATELLATM RABIES II, have been reported to
have better correlation with the RFFIT and FAVN test. For example, in comparative
testing with the RFFIT, the sensitivity and specificity of the PLATELLATM RABIES
II ELISA kit was reported to be approximately 95% (102). Additionally, a competitive
ELISA has been developed, using highly-purified G as the target antigen, that reportedly
will bind neutralizing-related antibodies (103). In the development of this competitive
ELISA, 4350 canine serum samples were tested in a comparative study using the FAVN
test where the results indicated that there were no false positives or negatives and that
there was a correlation between serological titre results as determined by the ELISA
and FAVN test of 96.2% (103). The remaining 3.8% of serum samples tested had titres
above the level of 8.0 IU/mL when assayed by both testing methods, and the serological
titre results from both tests were more divergent at high titre levels.

4.4 Measuring cell-mediated immunity

Assays to measure a cell-mediated immune response are usually used for research
purposes only because they are more complicated to perform on a routine basis than
serological assays. Detection of a cell-mediated immune response is commonly achieved
by measuring an increase in lymphocyte proliferation using a [H3]thymidine assay.
Methodologies for [H3]thymidine assays are published elsewhere (36,73). Newer assays
have been developed to measure cell-mediated immunity that utilize cell-tracking dyes
in conjunction with flow cytometry and are able to quantify the response of specific
types of lymphocytes to rabies antigen (36,106).

13
 5. Innocuity and efficacy of
rabies biologicals

The development and widespread use of rabies biologicals prepared on cell culture
have dramatically increased the safety and efficacy of PEP (23). Failures of rabies PEP
have been reported in some patients in developing countries, but in most of these cases
some deviation was reported from the WHO recommended PEP protocol (25,28,57).
Generally, the reasons associated with ‘treatment’ failures (where there was a failure
to follow the correct PEP protocol) include: delays in seeking treatment; lack of,
or improper primary wound care; lack of, or improper administration of RIG;
suturing wounds without infiltrating with RIG, or poor quality rabies vaccines
(25,28,107). The number of ‘true’ PEP failures (where a patient died despite receiving the
correct PEP protocol in a timely manner) are very low when compared to the millions
of doses of CCVs administered globally each year (25,107). Short incubation periods
of less than one week have been reported in patients that have severe head wounds,
such as in patients that have sustained brachial-lexus injuries from dog bites (20).
In one paper that examined case records from 15 human rabies patients reported
worldwide, it was concluded that seven patients received PEP in a timely and appropriate
manner, and still died of rabies (28). The paper discusses potential reasons for these
failures, including the possibility that a small unidentified wound may have been
overlooked, that perhaps one or more of the patients may have had an underlying
immunosuppressive condition, that the biologicals used to treat these patients were of
low potency, or perhaps that the treatment protocols were misrepresented. In any case,
all of the treatment failures occurred after dog bites, and the paper stresses the fact that,
on rare occasions treatment failures may occur, even with CCVs and RIGs.

Local and systemic reactions have been recorded after the administration of CCVs in
clinical trials (30,31,32,69). These studies generally reported local reactions, including
pain, itchiness, redness and/or swelling at the site of injection in 35% to 45% of the
enrolled subjects. Common systemic reactions, which are usually reported in 10% to
15% of subjects include fever, myalgia, malaise, headaches, dizziness, hives and rash
(108).

14 WHO immunological basis for immunization series - Module 17: Rabies

 6. Future prospects

The CCVs currently recommended by WHO are among the most efficacious vaccines
to combat disease available in the world. Only a few human deaths have been reported
in the literature in cases when WHO-recommended PEP protocols were strictly
adhered to (25,28). In spite of this, rabies continues to kill tens of thousands of people
and cost billions of US dollars annually (15). Most of the burden of rabies is felt by
those living in poor countries that can least afford to provide adequate PEP treatment.
Unfortunately also, rabies continues to spread to previously rabies-free areas and to
cause human fatalities, for example, the recent introduction of canine rabies on to the
island of Bali (109).

Although clinical rabies is preventable, even after exposure, a lack of educational

awareness is one of the major reasons why humans exposed to bites from infected
animals do not seek proper treatment after exposure (29). The cost of rabies biologicals,
and the frequent necessity to travel long distances over extended periods to receive one
of the recommended WHO PEP regimens, are also deterrents for persons exposed
to rabid animals (110). Newer rabies biologicals, for example monoclonal antibodies
targeted against rabies virus antigens that are currently undergoing clinical trials,
or are being evaluated for clinical trials, will hopefully provide a wider global access
to passive immunization at a reduced cost (111,112). Reduced-dose regimens for PEP
and PrEP, that can be completed within one week, are currently under evaluation,
and may also provide a solution to reduce the expense for patients that cannot afford
to travel to clinics located outside their immediate area to receive multiple doses of
rabies vaccine over extended periods of time (113). Molecular techniques are providing
new concepts for the development of rabies vaccines, for example, subunit vaccines and
safe modified-live viral vaccines, that could reduce the number of doses required for
PEP and PrEP to one, and significantly lower the cost to protect an entire population
(26,114,115). In addition, diagnostic tests, such as the dRIT, that are inexpensive,
simple to use and capable of rapidly producing accurate data, will help facilitate
surveillance in many poor settings. Finally, it is only through the introduction and
embodiment of comprehensive rabies control strategies, including animal control
programmes, PEP and PrEP for humans, educational programmes and financial
commitment, and ultimately cooperation between public-health professionals,
research scientists, laboratory technologists, not-for-profit organizations and
government officials that rabies will eventually be controlled (13,19,116,117).

15
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The World Health Organization has provided
technical support to its Member States in the
field of vaccine-preventable diseases since
1975. The office carrying out this function
at WHO headquarters is the Department of
Immunization, Vaccines and Biologicals (IVB).
IVB’s mission is the achievement of a world
in which all people at risk are protected
against vaccine-preventable diseases.
The Department covers a range of activities
including research and development,
standard-setting, vaccine regulation and
quality, vaccine supply and immunization
financing, and immunization system
strengthening.
These activities are carried out by three
technical units: the Initiative for Vaccine
Research; the Quality, Safety and Standards
team; and the Expanded Programme on
Immunization.
The Initiative for Vaccine Research guides,
facilitates and provides a vision for worldwide
vaccine and immunization technology
research and development efforts. It focuses
on current and emerging diseases of global
public health importance, including pandemic
influenza. Its main activities cover: i ) research
and development of key candidate vaccines;
ii ) implementation research to promote
evidence-based decision-making on the
early introduction of new vaccines; and iii )
promotion of the development, evaluation
and future availability of HIV, tuberculosis
and malaria vaccines.
The Quality, Safety and Standards team
focuses on supporting the use of vaccines,
other biological products and immunization-
related equipment that meet current inter-
national norms and standards of quality
and safety. Activities cover: i ) setting norms
and standards and establishing reference
preparation materials; ii ) ensuring the use of
quality vaccines and immunization equipment
through prequalification activities and
strengthening national regulatory authorities;
and iii ) monitoring, assessing and responding
to immunization safety issues of global
concern.
The Expanded Programme on Immunization
focuses on maximizing access to high
quality immunization services, accelerating
disease control and linking to other health
interventions that can be delivered during
immunization contacts. Activities cover:
i ) immunization systems strengthening,
including expansion of immunization services
beyond the infant age group; ii ) accelerated
control of measles and maternal and
neonatal tetanus; iii ) introduction of new and
underutilized vaccines; iv ) vaccine supply
and immunization financing; and v ) disease
surveillance and immunization coverage
monitoring for tracking global progress.
The Director’s Office directs the work of
these units through oversight of immunization
programme policy, planning, coordination and
management. It also mobilizes resources and
carries out communication, advocacy and
media-related work.

Department of Immunization, Vaccines and Biologicals

Family and Community Health
World Health Organization
20, Avenue Appia
CH-1211 Geneva 27
Switzerland
E-mail: vaccines@who.int
Web site: http://www.who.int/immunization/en/
ISBN 978 92 4 150108 8