Generation of broadly neutralizing antibodies for the development of an enterovirus immunotherapy

HUMAN ENTEROVIRUSES POSE A SERIOUS THREAT TO GLOBAL PUBLIC HEALTH
Viruses belonging to the genus Enterovirus of the family of Picornaviridae are
major causes of serious disease in neonates and very young children worldwide.
The most well-known member of this family, poliovirus, is close to extinction
as a result of the World Health Organization (WHO) eradication campaigns.
However, the related enterovirus (EV) 71 has started causing massive outbreaks
in Asia with ten thousands of cases of severe neurologic disease and thousands
of cases of infant death. Another clinically relevant EV is EV68, which
recently caused a nationwide outbreak of severe respiratory disease in the USA.
In addition, coxsackie- and ECHO viruses are leading causes of sepsis-like
illness and aseptic meningitis. These EVs cause 10-15 million of symptomatic
infections in the USA each year, with around 75.000 cases of aseptic
meningitis. Enterovirus-associated disease and fatalities have brought a heavy
economic burden to regions with high prevalence. Furthermore, the emergence of
novel (sub)genotypes giving rise to serious outbreaks poses a global public
health threat that should not be neglected.

NO VACCINES OR ANTI-VIRAL DRUGS AGAINST ENTEROVIRUSES ARE AVAILABLE
The global polio eradication program has unintentionally overshadowed the
clinical relevance of other non-polio EVs. As a consequence, the development of
vaccines and therapies against these viruses has been lagging behind and even
in developed countries children die of non-polio EV infections. Intravenous
immunoglobulin (IVIg) administration is sometimes used to treat severe EV
infections, based on the protective effect of anti-EV neutralizing antibodies
(NAbs) expected to be present in the IVIg formulation. However, it is generally
not known whether titers of NAbs against the causative EV type are high enough
in the IVIg pools (derived from >1000 plasma donors). The massive outbreaks of
EV71 in Asia have triggered the development of EV71 vaccines and monoclonal
antibodies (MAbs). However, these approaches do not take into account the broad
range of clinically relevant EVs causing disease elsewhere. Also, vaccines
cannot be applied directly in newborns, the most vulnerable group for severe EV
infections, because of their incompletely developed immune system.

Cross-reactive epitopes as targets for therapy development
Neutralizing antibody responses are unavoidably elicited against the
immunologically dominant sites of EVs and such responses determine the
serotype. A significant proportion of the immunologically dominant determinants
in EVs are present in one of the structural proteins known as VP1. The epitopes
presented in the surface exposed loops of VP1 are highly diverse and
susceptible to antigenic drift which leads to the generation of escape mutants.
However, heterologous NAb responses have been observed in animal and human sera
and nAbs with a cross-neutralizing reactivity among polioviruses have been
isolated. This provides evidence of the presence of conserved epitopes among
EVs, which represent highly interesting targets for the development of broadly
reactive immunotherapies. Pepscan analyses have revealed the location of
several of those (linear) conserved epitopes.

The role of bNAbs in protection or clearance of heterologous infections has
been recognized in recent years and advances have been made in the development
of immunization strategies for the induction of bNAbs against human
immunodeficiency virus (HIV) and Influenza. These advances have triggered the
exploration of potent and safe immunotherapy regimens that could provide a
long-term remission for HIV. These same promising avenues prompted us to
isolate and characterize bNAbs against EVs from human individuals and to use
these as a scaffold for production of bNAbs for an EV immunotherapy.

Only a few studies in the EV research field on induction of bNAbs have been
described, of which the majority focuses on bNAbs among polioviruses of type 1,
2 and 3. Knowledge on bNAbs against enteroviruses and their target epitopes is
therefore very limited. Our approach of isolating EV bNAb producing B-cells
from human individuals is new and has not been published before.
Characterization of bNAbs and their antigenic sites will therefore contribute
to a better understanding of the humoral response against EVs and will be an
important step towards development of an EV immunotherapy.
Enteroviruses are highly prevalent, which is reflected by the high percentage
of seropositive children (above 5 years) and adults. In the Netherlands, for
instance, 80% of adults has high nAb titers against EV71. The IgG variable
regions of bNAbs against influenza and HIV, isolated from adult donors, were
shown to be highly mutated, indicating that these bNAbs underwent multiple
rounds of affinity maturation. Therefore, adults with a high probability of
repeated exposure to EVs (via frequent contact with children) are expected to
be a suitable source of bNAbs against EVs.

Primary Objective:
To isolate B-cells producing bNAbs against EVs from healthy human individuals
and to generate bNAbs from these B-cells for the development of a novel EV
immunotherapy.

Secondary Objective(s):
To further elucidate the humoral immune response to EVs by defining the potency
of epitopes we selected to capture B-cells to induce bNAbs against EV in humans

The design of this study will be a prospective observational study. Eligible
healthy adult volunteers (aged 18-65 years) will be informed on the study and
asked for informed consent. Healthy volunteers will be recruited at the
Academic Medical Center (AMC), Amsterdam via distribution of a poster,
presentation of the research proposal during work meetings and personal
communication. After written consent, study participants will be invited for a
one-time venous blood sampling (a maximum of 21 ml blood). Blood samples will
be labelled with blinded code numbers to prevent identification of the blood
donors. Blood samples will be further handled and stored at the AMC Department
of Medical Microbiology (headed by Prof. Dr. M.D. de Jong). Samples will be
stored under the above mentioned (anonymous) code for 5 years.

In total 21ml of blood will be sampled from each adult donor via a single
venupuncture by well-trained medical staff. We therefore do not expect a more
than negligible risk. Participation will consume a limited amount of time and
subjects might experience some level of discomfort during the blood withdrawal.
Isolation of bNAbs from these donors will be the first big step in the
development of an EV immunotherapy for the treatment of life threatening EV
infections in neonates and young children. Our approach would therefore greatly
improve patient care and thus reduce disease burden and child suffering
worldwide.