Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines

– The absence of viral contaminants in polio and HAV vaccines was established by immunological tests on the monkey kidney cell primary cultures before infection with the purified virus E

Sciences médicales / Medical sciences

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines

Nguyen Van Mana, Hoang Thuy Nguyenb, Huynh Thi Phuong Lienb, Nguyen Thu Vanb,

Nguyen Kim Giaob, Nguyen Minh Lienb, Nguyen Thanh Thuyb, Irene Duniad*, Jean Cohenc,

E Lucio Benedettid

aPoliomyelitis Vaccine Research and Production Center (POLIOVAC) , Hanoi, Viet Nam

bNational Institute of Hygiene and Epidemiology (NIHE), Hanoi, Viet Nam

cVirologie et immunologie moléculaire, institut national de la recherche agronomique, Jouy-en-Josas, France

dInstitut Jacques-Monod–CNRS–universités Paris-6 et Paris-7, France

Received 27 March 2001; accepted 30 May 2001

Communicated by Jean-Antoine Lepesant

Abstract – Gold immunolabeling combined with negative staining (GINS) provides a

valuable immunocytochemical approach that allows a direct ultrastructural definition of

all viral vaccine constituents that share common antigenic features with pathogenic viral

particles These results have implications for the development of viral vaccines since it

has been demonstrated that incomplete viral particles such as natural empty capsides

and Rotavirus-like particles lacking the infective genome are potential candidates for the

production of neutralizing antibodies Furthermore comparative results of the

applica-tion of GINS to either inactivated vaccines or unfixed samples provide direct evidence

that even after inactivation specific antigenic sites are still available for gold

immunola-beling © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS

gold immunolabeling / negative staining / Polio-virus / Japanese encephalitis virus / hepatitis viruses /

Rotavirus (RV) / Rotavirus-like particles (VLPs)

Résumé – Caractérisation Immunocytochimique de virus et de macromolécules

antigéniques dans les vaccins antiviraux.L’application combinée de la coloration

négative et de l’immunomarquage à l’or collọdal (GINS, pour ‘gold immunolabeling

combined with negative staining’), permet d’identifier à la fois les caractères structuraux

et les propriétés antigéniques de plusieurs vaccins à l’aide des anticorps spécifiques

L’application de cette méthode aux vaccins contre les virus de la polio et de l’hépatite A,

a mis en évidence la présence de très nombreuses capsides vides dans les préparations

vaccinales Ces structures ont une antigénicité élevée tout à fait comparable à celle des

particules virales complètes De même, l’étude de capsides de rotavirus dépourvues de

génome, produites par des systèmes recombinants, montre que ces pseudo-particules

virales ont une antigénicité élevée La méthode décrite est une technique relativement

rapide et simple, adaptée aux moyens des laboratoires de pays en voie de

développe-ment Nos résultats peuvent aussi contribuer au choix d’une stratégie dans la production

de vaccins, fondée sur l’isolement et la production de particules pseudo-virales,

dépourvues de génome, mais hautement antigéniques © 2001 Académie des

sciences/Éditions scientifiques et médicales Elsevier SAS

immunomarquage à l’or / coloration négative / virus de la polio / encéphalite japonaise / hépatite

A, hépatite B / virus : poliomyélite, rotavirus (RV) / particules pseudo-virales de rotavirus (VLPs)

*Correspondence and reprints.

E-mail address: dunia@ijm.jussieu.fr (I Dunia).

© 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS Tous droits réservés

S0764446901013609/FLA

Version abrégée

L’application conjointe de l’immunomarquage à l’or

collọdal et de la coloration négative (GINS), à l’étude

de plusieurs vaccins par microscopie électronique, a

permis d’identifier les caractères structuraux de

parti-cules virales et, simultanément, les propriétés

antigé-niques de constituants vaccinaux actifs

Dans les vaccins contre les virus de la polio et de

l’hépatite A, nous avons pu observer de nombreuses

capsides virales dépourvues de matériel génomique

Cependant, ces structures conservent leurs propriétés

antigéniques telles qu’on peut les déduire par l’intensité

du marquage obtenu avec la méthode décrite De

même, les particules pseudo-virales de rotavirus

pro-duites dans des systèmes recombinants et constituées

par l’assemblage in vitro de protéines de rotavirus, sont

intensément immunomarquées

L’application de cette méthode a mis en évidence

des différences structurales entre des vaccins de

l’hépatite B préparés de deux façons différentes : 1) le

vaccin purifié par l’isolement de l’antigène de surface

HBsAg à partir de sérum de porteurs sains du virus ;

2) le vaccin préparé en produisant l’antigène HBsAg

dans un système recombinant L’antigène HBsAg

d’origine humaine est caractérisé par la présence de

nombreuses particules sphériques de 20 nm de

diamètre et la présence de nombreux composants

tubulaires En revanche, l’antigène HBsAg préparé

dans un système recombinant ne contient que des

particules sphériques de 20 nm de diamètre Cette

différence morphologique et

probablement antigénique, est due à la composition protéique de chaque préparation Dans le premier cas (antigène d’origine humaine), la présence d’un com-posant protéique de 44 kDa (L), responsable de la formation de tubules, a été décrite Ce polypeptide est absent de la préparation d’antigène du système recom-binant Il a été également décrit que les particules sphériques du HBsAg obtenues dans un système recom-binant contiennent le récepteur de l’albumine sérique humaine polymérisée (protéine de 34 kDa) Nous avons

pu mettre en évidence, avec nos résultats d’immunomarquage, que l’antigène HBsAg d’origine humaine contient aussi ce récepteur En effet, le blo-cage préalable par l’incubation exhaustive de ces pré-parations avec l’albumine sérique humaine, réduit con-sidérablement l’immunomarquage de l’antigène d’origine humaine Ces résultats indiquent que la méth-ode utilisée est suffisamment sensible pour permettre l’étude qualitative de préparations vaccinales D’autre part, l’analyse du nombre de particules d’or associées aux différents composants peut donner des indications utiles sur l’efficacité du marquage et, par conséquent, sur l’immunogénicité de particules virales de chaque vaccin étudié en tenant compte des différents procédés

de préparation (inactivation par la chaleur, fixation faible, etc.) Finalement, l’utilisation de cette méthode permet un contrơle de qualité constant, économique et rapide des préparations vaccinales, et aussi la possibil-ité d’envisager la production d’un vaccin basé sur la préparation de particules dépourvues de génome mais caractérisées par une antigenicité spécifique et significative

1 Introduction

Viral diseases cause major community health problems,

particularly in developing countries Despite great progress

in our knowledge of fundamental and applied virology,

there are only few identified natural or chemotherapeutic

agents that are able to cure viral illnesses Until recently

anti-viral vaccines were almost the unique successful

resource for prevention and treatment of many viral

syn-dromes [1–3]

At a time when vaccine production and quality

assess-ment are to be further developed, it is useful to have

advanced electron microscopy (EM) methods that can test

both the antigenicity and the ultrastructural features of

vaccine preparations

Several EM techniques have been developed that could

help in the identification of viral particles in a crude

sample and in the visualization of interactions between

viral components and specific antibodies [4–10] Cryo-EM

has opened a new avenue for high-resolution studies of

the viral architecture and its interactions with specific

antibodies [11–12] However, this technique requires fully specialized laboratory facilities that are not easily avail-able in developing countries In our opinion, the com-bined application of gold immunolabeling and negative staining (GINS), represents a method of more general applicability and can provide valuable information con-cerning the relation between ultrastructure and immuno-logical features of viruses and viral vaccines Viral vac-cines include live attenuated vacvac-cines, killed virus vaccines, antigenic macromolecules produced in living organisms by active viral infections and recombinant sub-unit vaccines [2] The data presented in this report dem-onstrate that the application of GINS to virus and viral vaccines is suitable for the identification of the structural entities bearing the antigenic determinants which are selec-tively enriched during the purification procedure of the vaccine Although the purification and quality control of a vaccine preparation can be tested by various biochemical and/or immunological methods, GINS may have the advantage of providing a direct immunocytochemical

esti-mation of the antigenic particle population and at the

same time, of the presence of non-immunogenic

contami-nants

2 Materials and methods

2.1 Viral and vaccine preparations

– The Japanese encephalitis virus (JEV) vaccine was

pre-pared as described [13] Briefly, viral particles were

puri-fied from brain tissue of Swiss mice infected with human

JEV The virus was then inactivated by treatment with

1/4 000 formaldehyde for 40 h

– Poliovirus vaccine was prepared from monkey kidney

cell primary cultures infected with poliovirus, according

to the protocol developed in POLIOVAC-NIHE The

vac-cine preparation was also inactivated by 1/4 000

formal-dehyde for 40 h

– Hepatitis A virus (HAV) vaccine was prepared from

monkey kidney cell primary cultures infected with HAV

isolated from human carriers, according to the technique

of purification of the Laboratory of Hepatitis Virus of the

National Institute of Hygiene and Epidemiology (NIHE),

Hanoi, Vietnam The purified vaccine preparation was

inactivated with by 1/4 000 formaldehyde for 40 h

– Hepatitis B surface antigen (HBsAg) was purified from

asymptomatic human carriers but with a high HBsAg titer

as described in [14] The vaccine preparation was

inacti-vated by 1/4 000 formaldehyde for 40 h

– Recombinant HBsAg produced in Chinese hamster

ovary transfected cells (CHO) with the hepatitis B virus

recombinant plasmid [15], was a generous gift of Dr M.L

Michel, who has carried out SDS-polyacrylamide gel

elec-trophoresis and immunoblotting experiments to

caracter-ize the protein constituents of HBsAg preparations [15],

(Laboratory of HBV, Pasteur Institute, Paris)

– Rotavirus (RV) and Rotavirus-like particles (VLPs), were

prepared as described previously, [16–18] Rotavirus

preparations for GINS experiments were at times

inacti-vated by 0.2 % formaldehyde treatment for 10 min

– The absence of viral contaminants in polio and HAV

vaccines was established by immunological tests on the

monkey kidney cell primary cultures before infection with

the purified virus (Elisa essays using specific reference

antibodies raised against SV40, PPLO and foamy virus,

provided by WHO)

2.2 Antibodies

Immunolabeling was carried out using specific primary

antibodies:

– Affinity purified rabbit antibody raised against purified

JEV prepared by the Encephalitis Virus Laboratory of NIHE,

Hanoi, Vietnam

– Affinity purified rabbit antibodies raised against

poliovi-rus prepared in the Laboratory of Virology, POLIOVAC,

Hanoi, Vietnam

– Human sera against HAV obtained from the Laboratory

of Hepatitis Virus of NIHE, Hanoi, Vietnam, and also from

the Laboratory of Virology, Hôpital Saint Antoine, Paris,

France

– Affinity purified rabbit antibodies raised against HB viral envelope proteins prepared at the Laboratory of Hepatitis Virus of NIHE, Hanoi, Vietnam

– Affinity purified rabbit antibodies raised against HBsAg produced in CHO cells transfected with plasmid contain-ing the S gene and the pre-S region of HBV [15]

– Rabbit polyclonal antibodies raised against RV proteins which recognize VP2, VP4, VP6, VP7

– Monoclonal antibodies E22 and RV138 raised against the RV VP2and VP6proteins, respectively [19–20] The specificity of the antibodies raised against JEV and poliovirus was established by comparative Elisa essays using as reference specific antibodies against JEV provided

by Bikan, (Osaka, Japan) and specific antibodies against poliovirus provided by WHO

The specificity of the antibodies raised against Rotavirus

proteins was tested by immunoblotting experiments using

purified Rotavirus capside proteins produced in the

recom-binant system [19]

Immunoblotting experiments were also carried out for testing the specificity of the rabbit antibodies raised either against human HBsAg or HBsAg from CHO recombinant system [15]

2.3 Gold Immunolabeling and Negative Staining

The GINS method that we developed is derived from the single drop technique [21] and carried out as follows:

20 mL droplets of viral or vaccine preparations are placed on a clean parafilm surface Collodium–carbon coated grids are made hydrophilic by rinsing them with 0.01 % Bacitracin in water The grids still wet are put on top of sample droplets for 5–10 min to pick up the sample The grids are washed with 2–3 droplets of PBS and then allowed to float on a droplet of PBS-2 % bovine serum albumin (BSA) for 20 min to block non-specific antigenic sites Some samples of HBsAg were incubated with PBS complemented with 2 % of human serum albumin (HSA) instead of BSA, as blocking step The grids were then reacted with the specific first antibodies, diluted in PBS–BSA 0.5 %, or PBS–HSA 0.5 % for 20 min After careful washes with PBS–BSA 0.2 %, or PBS–HSA 0.5 %, grids were subsequently incubated for 20 min with protein

A conjugated to 5 or 10 nm gold particles (Dept Cell Biology, University of Utrecht, The Netherlands) This step was followed by several washes with PBS and rapid fixa-tion (5 min) with 0.1 % glutaraldehyde in aqueous solu-tion For monoclonal antibodies, before incubation with gold-labeled protein A we used as bridge antibodies, a

15 min incubation with affinity purified rabbit anti-mouse immunoglobulins, diluted 1:500 in PBS–BSA 0.5 % The grids were then thoroughly washed with a solution of 0.1 % ammonium acetate, and negative staining was car-ried out using a 1 % aqueous solution of uranyl acetate or uranyl formate pH 5.4 Ammonium molybdate or phos-photungstic acid both at 1 % and pH 7, were occasionally used but uranyl salts yielded better results During the procedure the grids were not allowed to dry and care was

taken to maintain them floating on the drop surfaces After

negative staining, the grids were dried slowly before obser

vation All procedures were performed at room

tempera-ture We carried out control experiments testing the

speci-ficity of the immunolabeling by treating the samples

directly with gold labeled protein A, without previous

incubation with specific antibodies Other control

experi-ments were carried out by incubating the samples with

non-specific antibodies

Specimens were examined with a JEOL EM 1010 and a Philips EM CM12 both EM working at 80 kV

2.4 Chemicals

Unless otherwise indicated, all chemicals used for this work were purchased from Sigma Chemicals Co (St Louis

MO, USA)

Figure 1 Poliovirus vaccine

preparation immunolabeled using

affinity purified rabbit antibodies

raised against poliovirus.

A Poliovirus vaccine preparation

showing the presence of 30-nm

intact round-shaped virions

mixed with particulate material.

Both components are

immunola-beled Arrow points to an empty

viral shell Arrow heads point to a

viral particle displaying features

of disassembly Bar: 40 nm.

B Gallery of empty viral shells

also immunolabeled Bar: 20 nm.

3 Results

In our investigation we selected different viral vaccines

that are commonly used for control and prevention of

serious viral diseases in many developing countries To test

the general applicability of GINS, we carried out labeling

experiments on viral particles of different sizes and

struc-tural organization such as Picornaviridae and Rotavirus In

the case of the HBV vaccine we studied by

immunolabel-ing the specific viral antigen HBsAg isolated from human

carriers during the various steps of purification We also

compared the HBsAg isolated from human carriers with

similar antigenic molecules constructed by transfection of

recombinant plasmids The main issue of our investigation

was to characterize the immunochemical features of viral

vaccine preparations using specific antibodies and sera

from human carriers Furthermore, in view of analysing

the capability of GINS to identify specific viral protein

domains, we studied RV and Rotavirus-like particles (VLPs)

using monoclonal antibodies raised against single viral

polypeptides [16–19]

3.1 Poliovirus

Poliovirus, genus Enterovirus, is a member of the

Picor-naviridae It consists of roughly spherical virions 24–30 nm

in diameter [12, 22] The intact particles viewed in the

vaccine preparation are intensely gold immunolabeled

(figure 1A) In addition, several empty shells (20–30 nm)

are found (figure 1B) The empty shells, as well as the viral

material at all stages of its degradation, are positively

tagged with gold immunolabeling using antibodies raised

against Polio virus particles (figure 1A, arrows and 1B) No

contaminant viral particles could be detected in any

vac-cine preparations

3.2 HAV vaccine

HAV hepatovirus is a member of the Picornaviridae

[23–24] The diameter of the viral particles ranges between

20–30 nm The viral proteins are assembled according to a

dodecahedral model in which a capsid surrounds the RNA

core [25] The vaccine preparation is composed of viral

particles (20–30 nm in diameter, figure 2) The virions are

tagged with gold labeled protein A when the sample was

previously incubated either with specific antibodies raised

against HAV or with HAV positive human serum

(figure 2B) The HAV vaccine preparation is also

charac-terized by many round or oval shells (20–22 nm in

diam-eter), that likely correspond to natural empty capsids

(NECs; figure 2A), [23] The empty shells are also gold

immunolabeled In addition, the vaccine HAV preparation

comprises desintegrated virions displaying a peripheral

protein shell partially stripped away from the core This

material is also gold immunolabeled and has certain

com-mon features with the ‘skullcaps’ (figure 2C), [23].

3.3 JEV vaccine

JEV belongs to the genus Flavivirus (family Flaviviridae).

Virions are spherical, 45–55 nm in diameter [22, 26] They

are characterized by a membranous envelope and a fine peplomer surrounding a spherical nucleocapsid with yet unknown symmetry The vaccine preparation contains many intact viral particles 45–50 nm in diameter which

are intensely gold immunolabeled (figures 3A and 3B).

Another component is represented by round-shaped empty

shells (30–40 nm) (figure 3C) Furthermore, small

particu-late entities which have structural features comparable to

fragments of the viral membrane envelope (figure 3A

arrows) are also immunolabeled

3.4 Hepatitis B viral vaccine (HBsAg from human serum

of HBV carriers)

The major constituent of the HB vaccine consists of

20–22-nm particles of roughly spherical shape (figure 4A).

Figure 2 HAV vaccine preparation immunolabeled using human

sera against HAV.

A HAV vaccine preparation comprises purified 20-nm round-shaped

empty viral capsides (arrows) which are immunolabeled using human sera against HAV Bar : 45 nm.

B Immunolabeled round-shaped 30-nm virion with a distinct outer

layer Bar: 20 nm.

C The disassembly of a viral particle generates platelets or ‘skullcaps’

(arrow head) Bar: 50 nm.

Tubular-shaped components (20 nm thick) of variable

lengths are also observed (figure 4B) All particulate

enti-ties are heavily gold immunolabeled by specific HBsAg

antibodies (Figures 4A and 4B) Some HBsAg preparations

from the sera of human carriers were examined before the

last run of the process of purification by gradient

centrifu-gation and found to contain aggregates of round-shaped

20-nm particles and tubular components (figure 4C).

Occasionally these clusters are associated to granular

material that is immunolabeled with antibodies raised

against human IGg (figure 4C, arrows).

We have examined an HBsAg preparation produced in

CHO cells [15] transfected with a recombinant plasmid

containing the S gene and the pre-S region of HBV This

preparation consists of an homogeneous population of 20–22-nm spherical particles and no tubular components

could be detected (figure 5) The particles are intensely

gold immunolabeled by anti-HBV antibodies, particularly rabbit polyclonal antibodies raised against the HBsAg

produced by transfection of the recombinant plasmid

(fig-ure 5) (15) When the preparation of HBsAg purified from human serum of HBV carriers is pre-incubated with HSA

as blocking agent instead of BSA, the gold immunolabel-ing, using the rabbit polyclonal antibody raised against

HbsAg, is dramatically reduced (figure 6A) This residual

labeling accounts for less than 10 % as compared to more than 80 % of labeled particles when BSA is used Reduc-tion of the gold immunolabeling of HBsAg-CHO particles was also apparent when the preparation is pre-incubated

with HSA (figure 6B).

3.5 RV and VLPs

Inactivated viral particles [12], purified from RV (family, Reoviridae) infected cultures, are 75 nm in diameter and look like a wheel with a defined smooth outer rim Using polyclonal antibodies which recognize several viral pro-teins (VP2, VP6and VP7), gold immunolabeling is detected either surrounding the periphery of the virus or at the viral

surface (figure 7A) The VLPs generated by the assembly of

VP2, VP6 and VP7 are 70 nm in diameter and have a

triple-layered organization (figure 7B) Gold

immuno-labeling with polyclonal antibodies appears as clusters of gold particles either around the periphery of VLPs or inside

the triple-layered structure (figure 7B) The average

num-ber of gold particles labeling the intact formaldehyde inactivated RV counted in>30 particles was of 13 gold particles per virion and 5 for triple-layered VLPs The VLPs comprising only VP2and VP6are round-shaped particles

of about 60 nm diameter displaying a double-layered

organization (figure 8A) The outer layer consists of a regular assembly of subunits (figure 8A) The inner layer appears as a rather uniform stratum (figures 8B–8D)

Frag-ments of double-layered VLPs are frequently found in these preparations and have semi-circular profiles charac-terized by the subunit organization of the uneven outer

layer wrapping the inner stratum (figures 8B–8D) When

the monoclonal antibody raised against VP6is used the subunits forming the outer layer appear intensely tagged

by gold particles (figure 8A) Conversely, gold

immunola-beling is mainly restricted to the inner layer when VLPs

VP2–VP6 are incubated with the monoclonal antibody raised against VP2(figure 8B, arrow) Figure 8C shows one

layered VLP intensely gold labeled with VP2monoclonal

antibody Figure 8D shows the cup-like aspect of the

double-layered broken VLP; the VP2monoclonal antibody

has access to the VLP inner layer (Figure 8D) The unfixed

double-layered VLPs (VP2-VP6), immunolabeled with the monoclonal anti-VP6were tagged by an average number

of 28 gold particles, whereas using the monoclonal anti-body anti-VP2, 7 gold particles were found associated with the viral inner layer, (the total number of VLPs counted in each case was 30)

Figure 3 JEV vaccine preparation immunolabeled using affinity

puri-fied rabbit antibody raised against JEV.

A The preparation is characterized by the presence of

45–50-nm-immunolabeled intact virions The JEV sample also comprises

par-ticulate entities of different shapes, likely resulting from the

degrada-tion of viral particles This material appears also gold immunolabeled

(arrows) Bar: 50 nm.

B Higher magnification of viral particles showing the outer

membra-nous shell and the dense core of the viral capsid The gold particles

are associated to the outer surface of virions Bar: 30 nm.

C Empty viral shell intensely labeled Bar: 20 nm.

4 Discussion

4.1 Ultrastructural features of the viral vaccine

constituents bearing the antigenic activity

In spite of the inherent pitfalls of any technique of direct

structural observation of biological specimens, the

nega-tive staining method for electron microscopy since its

development, has provided the most straightforward and

comprehensive information on morphology and design principles of viruses [21, cfr.27] The range of applicability

of the negative staining technique can be expanded by using a novel approach that combines gold immunolabel-ing and the electron negative contrast These complemen-tary techniques have already been successfully applied to several problems of fundamental and applied virology [4–8, 21, 28–29] The application of GINS to HAV, JEV and poliovirus vaccines provide evidence that in addition to a

Figure 4 HBsAg purified from

human sera of HBV carriers.

A The preparation consists of

round-shaped 20–22-nm particles which are intensely immunola-beled using antibodies raised against HBsAg-CHO Bar :

50 nm.

B Similar preparation displaying

the pleomorphic organization of HBsAg characterized by round-shaped particles and tubular structures both intensely immu-nolabeled using antibodies directed against HBsAg from human sera Bar: 80 nm.

C The sample has been

exam-ined before the last run of the process of purification by gradi-ent cgradi-entrifugation It comprises clusters of 20-nm particles, tubu-lar structures, mixed with a minor amount of granular material that appears immunogold labeled using an antibody directed against human IgG (arrows) Bar: 55 nm.

major population of intact virions, the preparations

con-tain empty shells and structural entities reflecting all stages

of disassembly of the viral particles The presence of

empty viral shells is a commun feature of purified

Picor-naviruses and other viruses (JEV), [30–31] More recently,

these particulate entities were called NECs for ‘natural

empty capsides’ [23] It is assumed that both infective

virions as well as empty viral shells are produced when the

viruses grow in tissue cultures [32] Empty shells [30] may

have different sedimentation coefficients and variable

pro-tein compositions in comparison to infective virions [23]

It has also been claimed that empty shells can be produced

during the processes of purification and inactivation of

viruses [23] When Poliovirus inactivation is carried out by

harsh heating (56 °C), the RNA genome is released from

the virion and the protein shells are simultaneously

con-verted to a different antigenicity unable to produce

neu-tralizing antibodies against the native infective virions [23,

33] However the native antigenicity of empty viral shells

can be preserved when mild conditions of purification and

inactivation are applied, as is the case of our vaccine preparations which are inactivated by low concentrations

of aldehydes Intact viruses (HAV, poliovirus and JEV) as well as the incomplete empty shells of our vaccine prepa-rations appear gold immunolabeled We may then assume that the purification and inactivation processes generated viral structural entities characterized by exposed antigenic sites that are able to interact with specific antibodies raised against the viral antigens These results need further inves-tigation with complementary immunoassays that could provide information on the immunogenicity of a vaccine preparations

4.2 Gold immunolabeling and negative staining applied to quality control of HBV vaccine

Previous observations on purified preparations of HBV

by negative staining, demonstrated the presence of 3 types

of particulate entities namely, the 42-nm double-shelled

‘Dane’ particles corresponding to the infectious HBV, a great number of 20-nm spherical particles and tubular

Figure 5 HBsAg produced in

CHO cells.

The sample comprises almost

exclusively round-shaped 20-nm

particles which are all

immunola-beled with rabbit polyclonal

anti-bodies raised against the

HBsAg-CHO Bar: 80 nm.

components of variable lengths [34, 35] A common

anti-gen, HBsAg, characterizes these different forms Our

HBsAg purified preparations lack Dane particles and

con-tain 20-nm spherical particles and tubular assemblies both

heavily immunolabeled using antibodies directed against

HBsAg The HBsAg 20-nm particles, produced in

trans-fected CHO cells [15], have almost the same

morphologi-cal features as the HBsAg 20-nm particles of our vaccine

preparation They appear also heavily immunolabeled

with the rabbit polyclonal antibodies raised against the

recombinant HBsAg However, the recombinant HBsAg

lacks the tubular forms that we frequently found in the

human preparation of HBV carriers The existence of

several morphological and immunochemical variants of

HBsAg isolated from different human donors [3, 36], from

squirrel and duck sera [34] is well documented

Morpho-logical differences are probably related to the variable

aminoacid sequence and protein composition of HBsAg

subunits [3] Gel electrophoresis and immunoblotting

experiments demonstrated that the HBsAg produced from

CHO clones contains polypeptides of 22 kDa (small, S)

and 34 kDa (middle, M) but lacks the large polypeptide (L)

of 44 kDa [15] It is assumed that the presence of the L polypeptide is responsible of the assembly of the tubular and filamentous structures [37, 38], therefore it is not surprising that in the recombinant HBsAg the tubular forms are absent

HBsAg particles generated by CHO cells carry HSA receptors associated to the 34-kDa polypeptide [15] The presence of the 34-kDa protein likely confers to HBsAg a strong immunoreactivity in humans

In our experiments, pre-incubation with HSA as block-ing agent instead of BSA of both HBsAg form human origin and HBsAg-CHO, showed that the gold immunolabeling

is dramatically reduced We might then assume that HSA interacts with a major species-specific antigenic determi-nant – the 34-kDa polypeptide – thus preventing further immunolabeling reaction with the polyclonal antibodies directed against human HBsAg The attraction of this approach is that one acquires by the application of GINS

an indirect but useful estimate of the presence of specific components of the vaccine structural entites

Figure 6 A HBsAg purified from

human sera of HBV carriers The sample has been pre-incubated with HSA as blocking agent instead of BSA, followed by immunogold labeling using anti-bodies directed against HBsAg from human sera Immunogold labeling of both round-shaped 20-nm particles and tubular struc-tures is negligible (arrow) Bar:

55 nm.

B HBsAg produced in CHO cells.

The sample compring clusters of 20-nm particles has been pre-incubated with HSA The gold immunolabeling using rabbit anti-bodies directed against HBsAg-CHO is reduced (arrows) Bar:

60 nm.

The use of GINS has also contributed to address the

problem of the presence in the HBsAg isolated from human

carriers of immuno-complexes comprising HBcAg and

HBeAg [8] During the purification procedure of HBsAg,

immuno-complexes comprising HBcAg and HBeAg are

progressively eliminated and the application of GINS,

using specific anti-IgGs, showed that only in semi-purified

preparations could be identified a negligible amount of

human immunoglobulins associated to HBsAg These

results suggest that GINS may be a suitable tool to

imple-ment the quality control of vaccine samples during

purifi-cation procedures

4.3 Resolution power and labeling efficiency (LE) of GINS applied to RV, VLPs and viral vaccines

Among icosahedral viruses, the RV structure has been thoroughly assessed by using cryo-microscopy and 3D reconstitution methods [12] Stable VLPs are produced by expression in insect tissue cultures of Bacculovirus recom-binants with gene coding sequences for RV capsid pro-teins [16–18] Our data concern VLPs comprising either

VP2, VP6and VP7(triple-layered) or VP2and VP6 (double-layered) GINS applied to these structures confirms the notion that VLPs lacking RNA genome are self-assembled

Figure 7 A Intact RV of 75 nm

diameter, purified from RV

infected cultures RV are gold

immunolabeled with polyclonal

antibodies raised against viral

pro-teins VP2–VP7 Bar: 40 nm.

B Triple-layered subunit

organi-zation of 75-nm VLP generated

by the assembly of VP2, VP6and

VP7 The three layers are

immu-nolabeled by polyclonal

antibod-ies raised against VP2-VP7 Bar:

35 nm.