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Antibodies against immature virions are not a discriminating factor for dengue disease severity.

Rodenhuis-Zybert IA, da Silva Voorham JM, Torres S, van de Pol D, Smit JM - PLoS Negl Trop Dis (2015)

Bottom Line: We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups.Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease.Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands.

ABSTRACT
Humoral immunity plays an important role in controlling dengue virus (DENV) infection. Antibodies (Abs) developed during primary infection protect against subsequent infection with the same dengue serotype, but can enhance disease following secondary infection with a heterologous serotype. A DENV virion has two surface proteins, envelope protein E and (pre)-membrane protein (pr)M, and inefficient cleavage of the prM protein during maturation of progeny virions leads to the secretion of immature and partially immature particles. Interestingly, we and others found that historically regarded non-infectious prM-containing DENV particles can become highly infectious in the presence of E- and prM-Abs. Accordingly, we hypothesized that these virions contribute to the exacerbation of disease during secondary infection. Here, we tested this hypothesis and investigated the ability of acute sera of 30 DENV2-infected patients with different grades of disease severity, to bind, neutralize and/or enhance immature DENV2. We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups. Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease. Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis.

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Related in: MedlinePlus

Immature particles play a significant role in ADE of std DENV2 elicited by all three severity groups of immune sera.Non-treated P388D1 cells and cells treated with furin inhibitor (FI) were infected with immature (A) and std (B) DENV2 at MOG 500 in the absence or presence of pooled immune sera at 6400x dilution. Virus production was detected as described in the legend to Fig 3. The error bars represent SEM derived from at least two separate experiments performed in triplicate. Mann-Whitney test was used to determine significance difference between the infection with and without FI; * = P < 0.05, (n.d.) denotes ‘‘not detectable”.
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pntd.0003564.g004: Immature particles play a significant role in ADE of std DENV2 elicited by all three severity groups of immune sera.Non-treated P388D1 cells and cells treated with furin inhibitor (FI) were infected with immature (A) and std (B) DENV2 at MOG 500 in the absence or presence of pooled immune sera at 6400x dilution. Virus production was detected as described in the legend to Fig 3. The error bars represent SEM derived from at least two separate experiments performed in triplicate. Mann-Whitney test was used to determine significance difference between the infection with and without FI; * = P < 0.05, (n.d.) denotes ‘‘not detectable”.

Mentions: It is known that the structure of a virion influences the ability of an antibody to neutralize or enhance infectivity [52–55]. Yet, the structure of DENV particles during acute infection is unknown. We therefore attempted to dissect how many prM-containing particles circulate in the blood of DENV-infected humans. To this end, the acute serum samples were added to anti-prM Ab-coated ELISA plates and the extent of virus binding was quantified by qPCR. Unfortunately, despite the high viremia titers in acute sera, the numbers of ELISA captured virions were insufficient to reach the threshold necessary for reliable qPCR measurements. Therefore we used an alternative approach and instead of evaluating the number of prM-containing particles, we now questioned to what extent (partially) immature particles contribute to the ADE profile measured with std DENV preparations. For this approach we take advantage of the fact that antibody-opsonized immature viruses require enzymatic activity of furin in host cells to become infectious [31–33]. The ADE properties were therefore measured in P388D1 cells, treated with decRRVKR-CMK, furin specific. We have used this approach before with monoclonal Abs and found up to 10-fold reduction in viral infectivity following infection of FI-treated cells with Ab-std DENV2 complexes [32, 34]. Prior to the experiment, we first confirmed that the infectivity of immature particles opsonized with polyclonal sera is dependent on the activity of cellular furin during viral entry (Fig 4A). The half-life of FI is approximately 4–8 hours in aqueous solution and therefore it is expected that the compound will not interfere with the maturation of newly assembled virions within infected cells. Indeed, under the conditions used, FI did not affect maturation of newly assembled virions, confirming the short lived nature of the inhibitor (control bars in Fig 4A and 4B). Importantly, inhibition of furin activity in target cells abolished infectivity of immature virions opsonized with immune sera, substantiating that maturation upon entry is a prerequisite for rendering prM DENV-immune complexes infectious. Next we, the ADE properties of std DENV2 opsonized with pooled sera from DF, DHF and DSS patients was tested at a 6400x sera dilution, the dilution that yielded the highest power of enhancement in non-treated cells. Fig 4B shows a significant reduction in viral infectivity in cells treated with FI, indicating that ADE is also caused by immature particles present within the standard virus preparations. Contrary to our hypothesis however, the relative contribution of immature virions and antibodies enhancing their infection did not vary between the different disease severity groups.


Antibodies against immature virions are not a discriminating factor for dengue disease severity.

Rodenhuis-Zybert IA, da Silva Voorham JM, Torres S, van de Pol D, Smit JM - PLoS Negl Trop Dis (2015)

Immature particles play a significant role in ADE of std DENV2 elicited by all three severity groups of immune sera.Non-treated P388D1 cells and cells treated with furin inhibitor (FI) were infected with immature (A) and std (B) DENV2 at MOG 500 in the absence or presence of pooled immune sera at 6400x dilution. Virus production was detected as described in the legend to Fig 3. The error bars represent SEM derived from at least two separate experiments performed in triplicate. Mann-Whitney test was used to determine significance difference between the infection with and without FI; * = P < 0.05, (n.d.) denotes ‘‘not detectable”.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356584&req=5

pntd.0003564.g004: Immature particles play a significant role in ADE of std DENV2 elicited by all three severity groups of immune sera.Non-treated P388D1 cells and cells treated with furin inhibitor (FI) were infected with immature (A) and std (B) DENV2 at MOG 500 in the absence or presence of pooled immune sera at 6400x dilution. Virus production was detected as described in the legend to Fig 3. The error bars represent SEM derived from at least two separate experiments performed in triplicate. Mann-Whitney test was used to determine significance difference between the infection with and without FI; * = P < 0.05, (n.d.) denotes ‘‘not detectable”.
Mentions: It is known that the structure of a virion influences the ability of an antibody to neutralize or enhance infectivity [52–55]. Yet, the structure of DENV particles during acute infection is unknown. We therefore attempted to dissect how many prM-containing particles circulate in the blood of DENV-infected humans. To this end, the acute serum samples were added to anti-prM Ab-coated ELISA plates and the extent of virus binding was quantified by qPCR. Unfortunately, despite the high viremia titers in acute sera, the numbers of ELISA captured virions were insufficient to reach the threshold necessary for reliable qPCR measurements. Therefore we used an alternative approach and instead of evaluating the number of prM-containing particles, we now questioned to what extent (partially) immature particles contribute to the ADE profile measured with std DENV preparations. For this approach we take advantage of the fact that antibody-opsonized immature viruses require enzymatic activity of furin in host cells to become infectious [31–33]. The ADE properties were therefore measured in P388D1 cells, treated with decRRVKR-CMK, furin specific. We have used this approach before with monoclonal Abs and found up to 10-fold reduction in viral infectivity following infection of FI-treated cells with Ab-std DENV2 complexes [32, 34]. Prior to the experiment, we first confirmed that the infectivity of immature particles opsonized with polyclonal sera is dependent on the activity of cellular furin during viral entry (Fig 4A). The half-life of FI is approximately 4–8 hours in aqueous solution and therefore it is expected that the compound will not interfere with the maturation of newly assembled virions within infected cells. Indeed, under the conditions used, FI did not affect maturation of newly assembled virions, confirming the short lived nature of the inhibitor (control bars in Fig 4A and 4B). Importantly, inhibition of furin activity in target cells abolished infectivity of immature virions opsonized with immune sera, substantiating that maturation upon entry is a prerequisite for rendering prM DENV-immune complexes infectious. Next we, the ADE properties of std DENV2 opsonized with pooled sera from DF, DHF and DSS patients was tested at a 6400x sera dilution, the dilution that yielded the highest power of enhancement in non-treated cells. Fig 4B shows a significant reduction in viral infectivity in cells treated with FI, indicating that ADE is also caused by immature particles present within the standard virus preparations. Contrary to our hypothesis however, the relative contribution of immature virions and antibodies enhancing their infection did not vary between the different disease severity groups.

Bottom Line: We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups.Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease.Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands.

ABSTRACT
Humoral immunity plays an important role in controlling dengue virus (DENV) infection. Antibodies (Abs) developed during primary infection protect against subsequent infection with the same dengue serotype, but can enhance disease following secondary infection with a heterologous serotype. A DENV virion has two surface proteins, envelope protein E and (pre)-membrane protein (pr)M, and inefficient cleavage of the prM protein during maturation of progeny virions leads to the secretion of immature and partially immature particles. Interestingly, we and others found that historically regarded non-infectious prM-containing DENV particles can become highly infectious in the presence of E- and prM-Abs. Accordingly, we hypothesized that these virions contribute to the exacerbation of disease during secondary infection. Here, we tested this hypothesis and investigated the ability of acute sera of 30 DENV2-infected patients with different grades of disease severity, to bind, neutralize and/or enhance immature DENV2. We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups. Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease. Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis.

Show MeSH
Related in: MedlinePlus