Impact Objectives• Use the RAPINA system to improve the standard of laboratory-based rabies
surveillance in the Philippines and beyond• Further utlilise the RAPINA system to understand VNA levels in HIV-positive
patients so as to tailor their treatment accordingly• Ultimately put an end to rabies once and for all even in rabies endemic countries

Leading the charge
against rabies
Below Professor Akira Nishizono introduces RAPINA, an innovative new
system his team has developed that will benefit all rabies endemic countries
Could you begin
with an introductionto yourself?
I graduatedfrom Oita
Medical University in Japan in 1985,
and subsequently worked as an internal
medicine physician and then began
engaging in the virology researchfield. After obtaining my PhD, I began
researching rabies and I am now Professor
in the Department of Microbiology
at Oita University. My main theme iscomprehensive research on rabies;
including virology, epidemiology, diagnosis,prevention and therapeutics.
What interests you most about this field?
Over the last 50 years, Japan has been arabies-free country. However, the diseaseremains endemic in most other countries.
It is one of the typical neglected tropicalinfectious diseases (NTDs). Unfortunately,
there are few rabies researchers in thismedical field in Japan. Therefore, I feel I
must fulfil my mission to engage in researchof this historic and important zoonotic
disease.
Rabies is still incurable and leads to death.
Until now, there have been no effective ways
to treat the disease once symptoms appear.
I want to develop therapeutic treatments
against symptomatic rabies. That is my key
motivation for continuing rabies research.

Can you explain more about your studyinto the effectiveness of the improved rapid
neutralising antibody detection system(RAPINA)?
We demonstrated a novel, fast andeasy test – RAPINA – as an alternative
to virus-neutralising antibodies (VNA)
determination assays. The test is based on
the principle of immunochromatography
combined with virus neutralisation(binding), both using the inactivated rabies
virus as antigen and using two types ofanti-G monoclonal antibody. The RAPINA
test has good positive and negative
predictive values corresponding to a VNA
level of 0.5 IU/mL, as recommended by theWorld Health Organization (WHO) andthe World Organization for Animal Health(OIE). The RAPINA test can be used oneither a large or small number of samples,allowing VNA to be predicted at any time orfor any purpose.
Does RAPINA have any additional uses?
In vaccine clinics RAPINA can also beutilised to evaluate the effect of a vaccine
in immune-compromised hosts, including
HIV-positive patients. The number of CD4+
T cells is a crucial factor for inducing alasting immune response after the rabiesvaccine. When changing the regimen
or considering the booster vaccination
schedule for HIV-positive patients, it may
be important to know the precise level of
VNA in these patients.

Can you explain the importance of yourwork in real-world terms?
Current rabies prevention and control
strategies consist of different methods,
including wild and domestic animalvaccination programmes, animal birth control,
responsible pet ownership, rabies education
and awareness, and cooperating with medical
and veterinary approaches as the ‘One Health’
concept. Generally, the mass vaccination ofdogs against RABV is a highly rational strategy
for interrupting the natural transmission cycleof urban rabies from the veterinary side andproper post-exposure prophylaxis (PEP) evenafter being bitten by a rabid animal, and is
one of the most effective tools for preventing
rabies from the medical side. However, theabove-mentioned effective strategies forrabies elimination have been hampered byseveral factors, including inferior priorityof political decision-making and economic
issues. If user-friendly tools were available inresource-limited countries or districts, rabies
control programmes could easily be achievedin the future.
Can you discuss the work you have been
engaged with in the Philippines?
We have launched an international
collaborative project between Japan and thePhilippines to improve laboratory-basedrabies surveillance in the Philippines insupport of rabies elimination efforts. Beyond
this, our work will benefit all rabies endemic
countries and districts, particularly thoseresource-limited areas in Asia and Africa.
www.impact.pub 39

Rabies elimination efforts
Collaborative work led by Oita University in Japan, is helping to move the world
one step closer to the eradication of rabies

Despite Japan being free of rabies for 50years now, the disease remains endemic
in most countries. This is because thesecountries often do not have sufficient funds
or the infrastructure required to employ theapproved gold standard for the definitive
diagnosis of the disease. The disease isextremely serious, infecting the brain andnerves and causing inflammation. Whatis more, once symptoms have appeared,
the prognosis is nearly always death.
More research surrounding the disease isrequired in order to successfully diagnose
and ultimately combat it.
Research led by Chief Investigator Professor
Akira Nishizono at Oita University in Japan,
is seeking to ultimately combat the ongoingissue of endemic rabies using a new
method – RAPINA. The novel approach has
been specifically developed to quickly, easily
and efficiently determine viral neutralising
antibody (VNA) levels. This is crucial
because rapidly and easily determining theVNA level against the rabies virus is key
to evaluating the protective immunity ofhumans and dogs, and also for assisting inthe strategy of eradication programmes.
Nishizono is Professor in the University’s
Department of Microbiology and specialises
in comprehensive rabies research,encompassing virology, epidemiology,diagnosis, prevention and therapeutics.
Using RAPINA, Nishizono and his team areworking to improve laboratory-based rabies
surveillance in the Philippines.
CRUCIAL SUPPORT
Nishizono’s research is funded by: theJapan Agency for Medical Research
40 www.impact.pub

and Development; Japan International
Cooperation Agency (JICA); Japan Society
for the Promotion of Science; and USJapan Cooperative Medical Sciences
Programme. Nishizono explains more:‘The official request for ‘Development of an
improved laboratory surveillance systemfor rabies elimination in the Philippines’
was submitted to the Japanese Government
from the Government of the Philippines forpossible JICA assistance under its Scienceand Technology Research Partnership for
Sustainable Development (SATREPS).
This promotes international joint-researchstudies supported by a grant from JICA and
the Japan Agency for Medical Research andDevelopment (AMED).’

assays. The determination of the VNA
response following immunisation againstrabies is an indicator of how effective thevaccine has been. Existing systems havedrawbacks in terms of their speed and cost,in addition to other factors. Nishizono
explains more: ‘To measure the level of VNA
in human and animal serum, functional
assays RFFIT or FAVN are internationallyacceptable at the laboratory level,’ he states.
‘However, these assays are time-consuming(it requires about one and half days),expensive (requiring cell culture, fluorescent
microscope, FITC-conjugated reagents),
require the live rabies virus and can be
performed only in restricted referencelaboratories with biosafety level three.’

The research underway is highlycollaborative, with involvement from anumber of key participants, including:
Beatriz Quiambao of the ResearchInstitute for Tropical Medicine, ClinicalResearch Division in the Philippines;
and Mary Elizabeth Miranda of the Field
Epidemiology Training Programme Alumni
Foundation, Inc. in the Philippines.
Nishizono explains why collaboration is so
important to the research: ‘Collaboration
is key because there are no longer rabies
cases in humans and animals in Japan,
and what strongly promotes our project is
intimate collaboration with researchers andhealth sector personnel in rabies endemic
countries.’

Indeed, the only test for the prediction ofVNA is ELISA. Although this is cheaper,
safer and easier to perform than the virusneutralisation test, its reliability depends
on the quality of the coating antigen andthe specific secondary antibody against
the species whose serum samples are tobe tested. Furthermore, this test has ahigh-cost in many developing countries
where VNA testing is needed. ‘It does notprovide a perfect alternative to the virusneutralisation test,’ Nishizono confirms.

A NOVEL ALTERNATIVERAPINA is an improved rapid neutralising
antibody detection system that the teamhas developed with a view to providing analternative to existing VNA determination

PUTTING RAPINA TO THE TESTEnter RAPINA, a novel, faster, easier andmore cost-effective test that can be used as
an alternative to VNA determination assays.
It works by using immunochromatography
combined with virus neutralisation, and uses
the inactivated rabies virus as antigen andtwo types of anti-G monoclonal antibody.
‘The advantages of the RAPINA test are
low costs for laboratories in terms of bothCollaboration is key because there are no longerrabies cases in humans and animals in Japan,and what strongly promotes our project is intimate
collaboration with researchers and health sector
personnel in rabies endemic countries

running cost (reagents, FITC-conjugates) and
fixed costs (expensive equipment including
fluorescent microscope, spaces including
BSL-3, personnel),’ highlights Nishizono.
‘Secondly, the RAPINA test is less time
consuming, taking approximately two hours.
Thirdly, the RAPINA test can predict the VNA
level in serum from humans or any kind ofanimal including dogs but not mice, because
there is no need to use species-specificsecondary antibody.’ What is more, the test
can be used in laboratories where a large
number of samples are screened.
In contrast, RFFIT/FAVN works by diluting
serum samples and mixing them in vitro
with the rabies virus before BHK-21 cells
are added. Following a 24-hour period ofincubation, cells are released by trypsin
treatment, fixed and permeabilised witha p-formaldehyde solution, and stainedwith a rabies virus nucleocapsid protein-

The RAPINA test is both faster and more cost effective
than previous rabies test systems

specific antibody conjugate. Next, thepercentage of virus infection inhibition
caused by specific antibodies present in
the serum are evaluated in a fluorescent
microscope. Using a reference serum, acorrelation curve between the IU/ml content
and the percentage of infective inhibitionis established and the VNA titers of serum
samples are obtained by extrapolation.
ENCOURAGING FINDINGSThe team has reported the performanceof RAPINA for evaluating the vaccination
status of humans and dogs by conducting
a multicentre comparison in Asia. The
researchers discovered that, comparedto the VNA level determined by standard
neutralising tests, the positive and negative
predicted values and concordance ratioof RAPINA were highly homologous and,
importantly, reproducible among different
laboratories.

Project Insights
FUNDINGJapan Agency for Medical Researchand Development • J apan International
Cooperation Agency (JICA) • Japan
Society for the Promotion of Science• US-Japan Cooperative Medical
Sciences Programme
COLLABORATORS
Beatriz Quiambao – Research Institutefor Tropical Medicine, Clinical Research
Division, Philippines • Mary Elizabeth
Miranda – Field Epidemiology Training
Programme Alumni Foundation, Inc.
Philippines
CONTACT DETAILS
Akira Nishizono
Project CoordinatorT: +81 975865710
E: a24zono@oita-u.ac.jp
W: www.med.oita-u.ac.jp/biseibut/
PROJECT COORDINATOR BIO
Akira Nishizono MD, PhD is Professorof Department of Microbiology, Oita
University, Japan. His specialisedarea is the comprehensive research ofrabies, including virology, epidemiology,diagnosis, prevention and therapeutics.
His newly launched international
collaborative project is to improvelaboratory-based rabies surveillancein the Philippines in support of rabies
elimination efforts.

In addition to concluding that RAPINA
will be an effective tool for monitoringvaccination coverage in resource-limited
countries, the researchers believe RAPINA
has the potential to be adapted to determine
neutralising antibody level in otherinfectious diseases. Looking ahead, theresearchers intend to continue their focus
and explore effective therapeutics against
systematic rabies patients. l
www.impact.pub 41